A Controlled and Reproducible Pathway to Dye-Tagged

Langmuir 2008, 24, 2277-2280

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A Controlled and Reproducible Pathway to Dye-Tagged, Encapsulated Silver Nanoparticles as Substrates for SERS Multiplexing Leif O. Brown* and Stephen K. Doorn Chemical Sciences and Engineering, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 ReceiVed December 10, 2007. In Final Form: January 20, 2008 Silver nanoparticles tagged with dyes and encapsulated within a silica layer, offer a convenient potential substrate for performing multiplexed surface-enhanced Raman scattering (SERS) analysis. In contrast to our earlier work with gold particles, aggregation of silver particles is found to be mostly independent of dye addition, allowing for a reproducible preparation in which aggregation is actively induced by the addition of NaCl. Separating the aggregation step eliminates competitive binding between the dyes and silica-coating reagents, enabling the efficient use of a wide variety of weakly binding dyes to conveniently generate robust, high-intensity SERS substrates at a variety of excitation frequencies.

The use of surface-enhanced Raman scattering (SERS) as an alternative technique to fluorescence for sensitive multiplexed detection in biological samples is of growing interest.1 In particular, it is widely understood that SERS is capable of the sensitivity necessary to compete with fluorescence,2,3 while offering the distinct advantage of very narrow, separable, spectral line widths and fingerprints. For many purposes, it would be advantageous to employ the SERS substrate in the form of gold or silver nanoparticles to give a detection agent that is transportable by solution. Such a substrate could, for example, replace the fluorescent-tagged beads used in flow cytometry to give a method of detecting analytes in flow using Raman spectroscopy. Drawing on silica-coating work pioneered by Liz-Marza´n,4 Natan, Nie, and others5 described dye-tagged, silica-coated gold nanoparticles as SERS substrates. Typically, dyes that bind strongly to gold were used, such as thiols, and isothiocyanates. These dyes gave access to a variety of spectral signatures, while the SiO2 coating protected both the metal and the dyes from chemical reactivity in analyte samples. Competition between dye tagging and silica coating limits the ability to both use these particles with a larger range of dye molecules and to significantly increase SERS intensities. Recently, we showed that SERS intensities from dye-tagged, silica-coated gold nanoparticles can be optimized by adjustment of the amounts of silane and tag used in the coating reactions.6 While this resulted in strong SERS intensities, it relied on the use of strongly binding dye molecules. Work by Gong5c demonstrated that competition between tagging and silica coating could be eliminated by condensing * To whom correspondence should be addressed. E-mail: [email protected]. (1) See, for example: (a) Sun, L.; Yu, C.; Irudayaraj, J. Anal. Chem. 2007, 79, 3981-3988. (b) Jun, B.-H.; Kim, J.-H.; Park, H.; Kim, J.-S.; Yu, K.-Y.; Lee, S.-M.; Choi, H.; Kwak, S.-Y.; Kim, Y.-K.; Jeong, D. H.; Cho, M.-H.; Lee, Y.-S. J. Comb. Chem. 2007, 9, 237-244. (2) (a) Kneipp, J.; Kneipp, H; Rice, W. L.; Kneipp, K. Anal. Chem. 2005, 77, 2381-2385. (b) Vo-Dinh, T. Trends Anal. Chem. 1998, 17, 557-582. (3) Vo-Dinh, T.; Yan, F.; Wabuyele, M. B. J. Raman. Spectrosc. 2005, 36, 640-647. (4) (a) Liz-Marza´n, L. M.; Giersig, M.; Mulvaney, P. Langmuir 1996, 12, 4329-4335. (b) Tunc, I.; Suzer, S.; Correa-Duarte, M. A.; Liz-Marza´n, L. M. J. Phys. Chem. B 2005, 109, 7597-7600. (5) (a) Doering, W. E.; Nie, S. Anal. Chem. 2003, 75, 6171-6176. (b) Mulvaney, S. P.; Musick, M. D.; Keating, C. D.; Natan, M. J. Langmuir 2003, 19, 47844790. (c) Gong, J.-L.; Jiang, J.-H.; Yang, H.-F.; Shen, G.-L.; Yu, R.-Q.; Ozaki, Y. Anal. Chim. Acta 2006, 564, 151-157. (6) Brown, L. O.; Doorn, S. K. Langmuir in press.

SiO2 directly onto the particle surface through direct reaction of tetraethylorthosilicate (TEOS) with citrate anions adsorbed to the metal surface. By avoiding the use of functionalized, strongly binding silanes, weakly binding SERS dyes could be employed, such as crystal violet and rhodamine 6G. Although they were successful in this objective, the SERS intensities obtained appear to be relatively low. One possible reason for this is that there was no attempt to actively induce aggregation of the nanoparticless rather, aggregation is limited to that intrinsically obtained through dye addition. In general, unaggregated nanoparticle systems lack “hot spots” between nanoparticles, and thus produce relatively weak SERS responses.7 Requirements for dye-tagged, coated nanoparticle aggregates for SERS multiplexing include (a) high SERS intensity; (b) convenient and reproducible preparation; (c) ability to use a wide range of SERS-active dyes. Convenience and reproducibility require a reliable source of nanoparticles and the ability to eliminate factors such as timed reagent additions and competing binding equilibria. To meet these requirements, here we present SERS substrates prepared from silver nanoparticles aggregated controllably and reproducibly by the addition of NaCl. This separate aggregation step eliminates competing surface interactions, allowing for the adsorption of a wide variety of dyes and for the optimization of SERS signals to high intensities, prior to encapsulation of the aggregates in a stabilizing silica coating. While our earlier work involved gold nanoparticles, the use of silver offers two major advantages. The SERS enhancement offered by silver particles is significantly greater than that of gold,8 and they can be tuned over a greater range of excitation frequencies. This latter point is due in part to the unaggregated plasmon resonance being situated at higher frequencys∼420 nm vs ∼520 nm for gold. We chose to prepare nanoparticles through a sodium citrate reduction of AgNO3.9 This simple procedure is both reliable and very reproducible, giving yellowgreen nanoparticles (λmax ) 414 nm,  ∼ 7 × 1010 mol-1 dm3 cm-1) that are stable at room temperature for periods in excess of 1 year. From electron microscopy (JEOL 3000F HRTEM, 300 keV), we found the nanoparticle size distribution to be 68 (7) Talley, C. E.; Jackson, J. B.; Oubre, C.; Grady, N. K.; Hollars, C. W.; Lane, S. M.; Huser, T. R.; Nordlander, P.; Halas, N. J. Nano Lett. 2005, 5, 1569-1574. (8) Aroca, R. F.; Alvarez-Puebla, R. A.; Pieczonka, N.; Sanchez-Cortez, S.; Garcia-Ramos, J. V. AdV. Colloid Interface Sci. 2005, 116, 45-61. (9) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391-3395.

10.1021/la703853e CCC: $40.75 © 2008 American Chemical Society Published on Web 02/16/2008

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Figure 1. Preparation of silica-coated, dye-tagged, silver nanoparticles: (i) addition of functionalized silane (MPTMS) to weakly stabilized particles, (ii) addition of SERS dye, (iii) controlled aggregation using NaCl, (iv) growth of SiO2 shell using sodium silicate.

( 17 nm, from which we derived a particle concentration of 110 pM, assuming complete reaction of the AgNO3. The particle solution can be further concentrated by careful evaporation. In direct contrast to the gold nanoparticles we studied previously,6 these silver nanoparticles once prepared can be used in glassware that has not been cleaned with aqua regia, a useful point of convenience. The dye tagging and silica coating procedure for the silver nanoparticles is illustrated in Figure 1. A thiol-functionalized silanes(3-mercaptopropyl)trimethoxysilane, MPTMSsis added to the nanoparticle to provide a foundation for growth of SiO2. The thiolated silane is chosen to provide strong adsorption without the binding equilibrium present in the more commonly used (3-aminopropyl)trimethoxysilane. By adding the silane first, any potential competition for the metal surface between the silane and the weakly binding dyes is eliminated. The SERS dye is then addedsin the gold process, this step causes rapid aggregation of the nanoparticles, yet dye addition in the silver process causes little to no aggregation (depending on the selected dye). This allows us to use NaCl as an aggregation agent in a third, separate step, resulting in a procedure that is both more reproducible and more controllable. (In contrast, the preparation of COIN-type particles deliberately uses high concentrations of dye species as an aggregation agent coupled with heat.10) The final step involves adding sodium silicate to grow the silica layer surrounding the dye-tagged aggregates. In a typical sequence, a silver nanoparticle solution (1 mL, 110 pM) is rapidly mixed with MPTMS (50 µL, 25 µM). After standing for 2 min, a solution of the chosen SERS dye (see discussion below) is added (typically 100 µL, 2.8 µM). Some minor aggregation may occur at this point and is usually complete within 1 to 2 min. Stronger controlled aggregation rate is then achieved through the addition of NaCl (quantity dependent on chosen dyestypically 20-120 µL, 170 mM). For controlled aggregation (see discussion below), the aggregation time is heavily dependent on the chosen dye and can vary from a few seconds to 30 min. During this time, measured SERS intensities will increase and then plateau. As a consequence, it is beneficial to allow the aggregation step several hours to complete and to verify (10) (a) Su, X.; Zhang, J.; Sun, L.; Koo, T.-W.; Chan, S.; Sundararajan, N.; Yamakawa, M.; Berlin, A. A. Nano Lett. 2005, 5, 49-54. (b) Sun, L.; Sung, K.-B.; Dentinger, C.; Lutz, B.; Nguyen, L.; Zhang, J.; Qin, H.; Yamakawa, M.; Cao, M.; Lu, Y.; Chmura, A. J.; Zhu, J.; Su, X.; Berlin, A. A.; Chan, S.; Knudsen, B. Nano Lett. 2007, 7, 351-356.

Figure 2. Silver nanoparticles tagged with different dyes. All spectra are offset 20 000 counts s-1 mW-1 and normalized to 110 pM unaggregated nanoparticle concentration, 1 mW excitation at the sample, and 1 s integration time. (a) Six dyes excited at 532 nm (DeltaNu Advantage 532 spectrometer, 3.4 mW). (b) Two dyes excited at 785 nm (Kaiser Optical Systems spectrometer, 18 mW - oxazine 170/72 mW - HITCI) and at 633 nm (DeltaNu Advantage 200A spectrometer, 1.7 mW). Samples prepared as described in the text, using the following amounts of 170 mM NaCl: Nile blue (20 µL), oxazine 1 (70 µL), oxazine 170 (20 µL), ethyl violet (40 µL), malachite green (60 µL), HITCI (120 µL).

the stability of the tagged material. Any observed decrease in the SERS intensity indicates the use of too much NaCl and the onset of an uncontrolled aggregation phase. As with the gold nanoparticles, a large variety of chromophoric dyes can be attached. Because the aggregation process is largely separated from the dye tagging, it is easier to utilize weakly binding dyes with the silver nanoparticlessfor gold nanoparticles, the adsorption equilibrium complicates the process of adding sufficient dye to give strong signals while simultaneously trying to control aggregation. Dyes we have successfully used include Nile blue, basic fuchsin, ethyl violet, rhodamine 800, rhodamine 590, thionin, crystal violet/malachite green, HITCI (1,1′,3,3,3′,3′hexamethyl-2,2′-indotricarbocyanine iodide), oxazine 170, and oxazine 1, in addition to strongly binding isothiocyanate dyes such as malachite green isothiocyanate, X-rhodamine-5(6)isothiocyanate, and tetramethylrhodamine isiothiocyanate. Some example spectra are shown in Figure 2. The SERS signal strength depends primarily on the degree of aggregation and the overlap between the dye absorption λmax and the laser excitation frequency. Thus, with the appropriate selection of long wavelength dyes, such as rhodamine 800 or oxazine 170, strong signals can be obtained with 532, 633, and 785 nm excitation. Some long-

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Figure 3. Effect of added NaCl on Nile blue-tagged (230 nM) Ag nanoparticles. (a) UV-vis-NIR spectra normalized for particle concentration (110 pM unaggregated) at 10 mm path lengthsinset normalized to λmax intensity. Instrumentation: Varian Cary 6000i, 5 mm path length, diluted samples. (b) SERS intensities (1640 rel. cm-1 peak) using 532 or 633 nm excitation, corrected for background, particle concentration (110 pM unaggregated), integration time, and excitation power at the sample. Error bars represent 1 standard deviation derived from 50 acquisitions.

wavelength dyes (e.g., HITCI) work best only with 785 nm excitation. Further, strong SERS is possible with 532 nm excitation simply because this wavelength is close to the plasmon resonance absorption of the nanoparticles. The effect of added NaCl on aggregation is illustrated by the UV-vis-NIR spectra displayed in Figure 3a, in which the Nile blue concentration is 230 nM. From 0 to 7.1 mM NaCl, the spectra show a broadening and red-shifting of the plasmon resonance (seen most clearly in the inset spectra), coupled with a decrease in its intensity, indicating a controlled aggregation. From 8.5 to 12 mM, the single-particle plasmon loses most of its remaining intensity and narrows as it continues to red-shifts aggregation through this region is less well defined. Greater NaCl concentrations result in visible flocculates and an electronic spectrum composed mostly of a scattering background. The aggregation effects seen in the electronic spectra correlate directly with the observed SERS intensities (Figure 3b). From 0 to 7.1 mM NaCl, there is a steady increase in the SERS intensities obtained from both 532 and 633 nm excitations. A characteristic of this controlled phase of aggregation is that the distributions of the SERS intensities remain fairly narrow (represented by the error bars in the figure), depicting a reasonably homogeneous sample. While flocculation due to higher NaCl concentrations localizes nanoparticles into aggregates of extremely high SERS intensity, the overall solution concentration of nanoparticles is lowered, resulting in lower average SERS intensities. As a consequence, the SERS intensity distributions become markedly

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Figure 4. Effect of Nile blue concentration on subsequently NaClaggregated (7.1 mM) Ag nanoparticles. (a) UV-vis-NIR spectra normalized for particle concentration (110 pM unaggregated) at 10 mm path length. (b) SERS intensities (1640 rel. cm-1 peak) due to 532 and 633 nm excitation as a function of Nile blue concentration. Error bars represent the standard deviation of intensities determined from 25 acquisitions. Intensities are corrected for background, particle concentration (110 pM, unaggregated), integration time, and excitation power at the sample.

wider as the NaCl concentration rises above 8.5 mM (see Supporting Information for examples). Using a NaCl concentration of 7.1 mM (which provides a strong, stable signal in the above analysis), the effect of varying the Nile blue concentration was investigated (Figure 4). It can be seen that the SERS intensities for the tagged nanoparticle aggregates rise gradually up to dye concentrations of ∼200250 nM (a concentration that provides at most ∼2000 dye molecules per metal particle), after which they fall with gradually increasing intensity distributions. As with the previous experiment, the results are determined by particle aggregation. At 7.1 mM NaCl and 230 nM Nile blue, an optimal state of aggregation is achieved from the large influence of NaCl combined with the lesser influence of Nile blue (which is itself a chloride salt)s resultant SERS intensities are in close agreement between the two experiments. As with excess NaCl, larger amounts of Nile blue cause overaggregation of the system with a resultant drop in SERS intensity. The overaggregation effect is less dramatic with the limited dye concentrations employed, as evidenced by the narrower intensity distributions. Other dyes can be stronger or weaker aggregation agents, as demonstrated by the relative amounts of NaCl added to achieve the SERS spectra in Figure 2b. Although the tagged nanoparticle aggregates are very stable, they are not chemically inert and must be protected in order to be of use in other chemical environments, such as multiplexing assays. For this reason, the aggregates are encapsulated in a

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Figure 5. TEM images (JEOL 3000F, 300 keV) of SiO2-coated, dye-tagged Ag nanoparticle aggregates after one week of exposure to sodium silicate. Note that exposure of the thin SiO2 layer to intensely focused beams results in rearrangement, stripping, and (occasionally) crystallization.

silica shell, using a process performed in an analogous fashion to that of the gold nanoparticles. Sodium silicate (120 µL per 1 mL of starting nanoparticles, 22 mM) is added to the dye- and silane-tagged silver particle aggregatessafter a period of one week, the aggregates are covered with a layer of SiO2 approximately 3-5 nm thick (Figure 5). Despite the small amount of silica present, the product aggregates tend to be more robust than the gold equivalent and are purified by centrifuge (14 500 × g, 35 min) repeated 6-7 times and replacing the supernatant each time. In many cases, the centrifuge steps are used to concentrate the aggregatesswe have done so up to 25 nM (unaggregated particle concentration) without observing any adverse effects, although the solutions become notably denser with a greater surface tension. Product aggregates in our possession for ∼1 year have shown no noticeable change in properties. In summary, silver nanoparticles can be used to produce silicacoated, dye-tagged SERS substrates with intensities significantly

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greater than the gold nanoparticle aggregates we reported recently. Although direct comparison is nontrivial, silver aggregates tagged with malachite green are typically a factor of ∼3-4 more intense with 633 nm excitation than their gold counterparts at the same particle concentration. In addition, silver aggregates offer greatly enhanced access to green (e.g., 532 nm) excitation wavelengths, while retaining the ability to work with red excitation. A major point of contrast between the two metal systems is that aggregation of the silver nanoparticles must be induced by the addition of NaCl. The separate aggregation step gives significantly enhanced control over the process and allows for greater reproducibility of SERS intensities. The separate aggregation step also eliminates competition between tagging and aggregation objectives, allowing for facile use of weakly binding dyes, thereby opening up a much larger selection pool of potential spectral tags. In contrast, the isothiocyanate functionalized dyes used in the gold system not only are costly and unstable as stock solutions but are commercially available for only a few basic dye structures. Coating the silver nanoparticle aggregates with a thin layer of SiO2 yields a very robust SERS substrate that is already being exploited in the development of Raman flow cytometry.11 Acknowledgment. This work was supported by LANL LDRD funding and also by NIH Grant Number NIH EB003824. Supporting Information Available: Expanded particle tagging procedure, and example data sets used to create Figure 3b. This material is available free of charge via the Internet at http://pubs.acs.org. LA703853E (11) Watson, D. A.; Brown, L. O.; Gaskill, D. F.; Naivar, M.; Graves, S. W.; Doorn, S. K.; Nolan, J. P. Cytometry A 2008, 73A, 119–128.