Analytica Chimica Acta 874 (2015) 49–53
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Quantitative surface-enhanced Raman measurements with embedded internal reference Yan Zhou, Rui Ding, Padmanabh Joshi, Peng Zhang * Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, United States
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A new methodology to perform quantitative SERS measurements of analytes using internal reference. SERS-based quantification of Toluidine Blue O as a proof-of-concept study. SERS-based quantification of melamine in milk.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 August 2014 Received in revised form 21 November 2014 Accepted 9 March 2015 Available online 11 March 2015
Analytical applications of SERS are often more associated with qualitative than quantitative analysis, because of the difficulty in obtaining quantitative SERS results. In this paper we introduce a new strategy to quantitatively measure the SERS signals of analytes based on Au-core/Ag-shell nanoparticles with embedded 4-aminothiophenol as the internal reference. Successful detections of two analytes, Toluidine Blue O in aqueous solution (detection limit of 0.1 mM) and melamine in milk (detection limit of 5 mM), are demonstrated. The improvement in the linear fitting illustrates that the use of internal reference significantly improves the accuracy of the quantitative SERS measurements. The successful detection of melamine in milk illustrates the versatility of this detection scheme for a wide variety of analytes. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Quantitative surface-enhanced Raman measurement Internal reference Aminothiophenol Melamine Core–shell nanostructure
1. Introduction
* Corresponding author. Tel.: +1 513 556 9222. E-mail address: [email protected] (P. Zhang). http://dx.doi.org/10.1016/j.aca.2015.03.016 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.
Surface-enhanced Raman spectroscopy (SERS) has proved to be a useful tool for diagnostic applications, and detection of explosives and bioanalytes [1–9]. Raman scattering of molecules, despite of being an inefficient process with very small crosssections, can be greatly enhanced by orders of magnitude if the
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Y. Zhou et al. / Analytica Chimica Acta 874 (2015) 49–53
molecules reside in the vicinity of some metal nanostructures, especially those of silver and gold [10,11]. The highly enhanced Raman signals have allowed SERS to be used as a tool in singlemolecule spectroscopy [12]. Due to the inherently narrow and fingerprint-specific spectra, SERS offers a greater degree of multiplexing capability, as compared to the more widely used fluorescence-based detection schemes [13,14]. However, analytical applications of SERS are often more associated with qualitative than quantitative analysis, because of the difficulty in obtaining quantitative SERS results. Quantitative SERS measurements are usually jeopardized by several factors, such as poor reproducibility of the SERS substrates, and variation in the adsorption of analyte to the substrate [15,16]. There have been efforts to address issues associated with the quantification aspect of SERS. For instance, a method was proposed to improve the accuracy of SERS measurements by using the SERS signal generated from a self-assembled monolayer (SAM) as the internal reference [17]. SAMs prevent chemisorption of the analyte onto the SERS-active surface and thus improve the reproducibility. Yet the detection limit and the dynamic range have been greatly compromised because of the distance between the analyte and SERS-active surface created by the SAM coating. A different group has introduced a so-called isotope edited internal standard method to improve the accuracy of quantitative SERS measurement [18,19]. An isotope-edited version of the analyte was used as the internal reference to minimize the difference in enhancement factors of the analyte and the reference. In practice, this approach is hindered by some intrinsic limitations. For instance, an isotope-edited counterpart is required for any given analyte of interest. Additionally, the SERS spectral features of the analyte and its isotope-edited internal standard have to be sufficiently different to facilitate independent measurement of their SERS intensities in a mixture. Since most molecules have very similar SERS spectral features with their isotope-edited counterparts, the types of intended analytes are limited. Competition between the analyte and its isotope-edited counterpart may cause extra uncertainty as both are directly attached to the same metal surface. Herein we report a new strategy for quantitative SERS measurements through an internal reference embedded inside the Au-core/Ag-shell nanoparticles, which serve as SERS substrates for analytes in the proximity. The scheme provides great flexibility in the choice of the internal references, which should be different from the analytes, and allows quantitative SERS measurements of the analytes in the solution. Two different types of analytes, Toluidine Blue O (TBO) and melamine, are studied in this paper. The TBO sensing is shown as a proof-of-concept study, while the melamine sensing in milk illustrates the practicality of this strategy in real applications.
2.2. Synthesis of citrate stabilized Au nanoparticles Gold nanoparticles of 20 nm were synthesized according to the procedures in the literature [20]. Briefly, in a 100-mL glass flask, 50 mL of a 0.01% aqueous HAuCl4 solution was heated to boil under magnetic stirring. Upon boiling, 1 mL of 1% sodium citrate was rapidly injected. Within seconds, the pale yellow solution turned into deep purple and quickly progressed to red. The colloid was kept boiling for 15 min to ensure complete reduction, before left to cool to room temperature. Excessive ions were removed from the solution by centrifugation, and the AuNPs were redispersed in 50 mL of DI water. The AuNPs solution was kept at 4 C until later use. 2.3. Synthesis of Au@ATP@Ag nanoparticles 10 mL of 4-ATP (0.1 mM in EtOH) was added to 10 mL as-synthesized citrate-stabilized AuNPs solution under vortex. The solution was incubated overnight at room temperature. Deposition of silver shell onto the 4-ATP-conjugated gold nanoparticles was similar to the method in the literature with minor modification [21]. Hydroquinone (300 mL, 10 mM in water) and AgNO3 (300 mL, 10 mM in water) were added in sequence to 1 mL Au@4-ATP solution under vigorous stirring. The mixture was incubated at room temperature for 12 h to allow for the completion of the reaction. The solution was then centrifuged for 10 min at 14,000 rpm to collect the precipitate, which was re-dispersed in 10 mL DI water until later use. 2.4. Synthesis of MDA–TBO conjugates In a typical reaction, MDA (23 mg, 0.01 mM) was dissolved in a mixture of 1 mL ethanol and 9 mL of 50 mM MES buffer (pH 6.5). Then, 20 mg EDC and 30 mg NHS was added in sequence to the solution. The activation reaction was allowed to proceed at room temperature for 20 min. TBO (31 mg, 0.01 mM) was then added to the solution under stirring. After stirring for 24 h, the final solution was diluted to 10 mM for later use. 2.5. Characterization UV–vis extinction spectra were recorded on a USB 4000 spectrophotometer (Ocean Optics, USA) using a 1-cm path length quartz cell at room temperature. TEM measurements were performed on a Biotwin 12 transmission electron microscope (FEI). TEM samples were prepared by depositing a drop of suspension containing nanoparticles on a formvar-covered carbon-coated copper grid (Electron Microscopy Sciences, PA) and letting it dried at room temperature.
2. Materials and methods
2.6. Raman measurement
2.1. Chemicals and materials
All samples for Raman measurement were prepared by adding a known amount of an analyte solution into Au@4-ATP@Ag solution. For TBO samples, MDA–TBO (10 mL, 10 mM) was added into 990 mL as-synthesized Au@4-ATP@Ag solution under magnetic stirring. By changing the concentration of MDA–TBO from 10 mM to 20, 30, 50, 60, 90 and 100 mM, the other six solutions were obtained for later use. Melamine was first dissolved in DI water to prepare a 10 mM stock solution. The melamine solution was next spiked into the milk purchased from a local grocery store, to obtain milk solutions containing 0.05, 0.1, 0.25, 0.5, 0.75 and 1 mM melamine, respectively. Then, samples with melamine concentration of 5, 10, 25, 50, 75, and 100 mM, respectively, were obtained by adding 100 mL of the melamine-containing milk solutions into 900 mL of as-synthesized Au@4-ATP@Ag solution. Raman measurements
4-Aminothiophenol (4-ATP), Toluidine Blue O (TBO), melamine, hydrogen tetrachloroaurate, 12-mercaptododecanoic acid (MDA), 2-(N-morpholino)ethanesulfonic acid (MES), sodium citrate, hydroquinone, sodium cyanide, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were obtained from Sigma–Aldrich. Milk was purchased in a local grocery store (Meijer). All chemicals, unless specified, were of reagent grade. Reagents and solvents were obtained commercially and used without further purification. All glassware were cleaned with freshly prepared aqua regia and rinsed thoroughly with DI water prior to use. All solutions were prepared using deionized (DI) water (18 MV-cm).
Y. Zhou et al. / Analytica Chimica Acta 874 (2015) 49–53
were done on a Renishaw inVia Raman microscope system. The aforementioned samples were dispersed thoroughly by sonication. A drop of the dispersion was transferred into a small quartz tube of 1 mm I.D. The tube was placed on the stage of the microscope for Raman measurements. The microscope objective has 10 magnification and 0.25 NA. Laser intensity at the samples was 11 mW using the 785 nm line of a diode laser for all measurements. Exposure time was 10 s for the TBO measurements and 20 s for melamine measurements. Between different Raman sessions, the 520.7 cm 1 peak of a silicon wafer was used to calibrate the spectrograph. Fig. S1 shows the Raman spectra of Au@4-ATP@Ag, solid TBO and solid melamine (Supporting information). 3. Results and discussion 3.1. Synthesis and characterization The design of the Au-core/Ag-shell nanoparticle with embedded internal reference is illustrated in Fig. 1. The method to synthesize internal reference-embedded Au-core/Ag-shell nanoparticles is similar to what was reported previously with minor modifications [21]. In brief, gold nanoparticles are first synthesized through thermal reduction of HAuCl4 by citrate according to the literature [20], and washed thoroughly before use. Probe molecules serving as internal reference are adsorbed to the gold nanoparticle surface, on top of which a silver layer is subsequently deposited. The probe molecule used in this work is 4-aminothiophenol (4-ATP). The formation of the Au-core/Ag-shell nanoparticles is confirmed by both UV–vis extinction spectroscopy and transmission electron microscopy (TEM), as shown in Fig. 2. The extinction spectrum of gold nanoparticles with 4-ATP attached (Au@4-ATP) includes the typical plasmon band of Au nanoparticles (520 nm). The spectrum of Au@4-ATP@Ag contains a broad shoulder band at 400 nm and a slightly blue-shifted Au plasmon band, both indicating the formation of the Ag-shell on the Au@4-ATP nanoparticles. The distinct core–shell structure can be observed in the TEM image (Fig. 2, Fig. S2). The Au@4-ATP@Ag nanoparticles are well dispersed in water and stable for weeks.
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TBO and MDA are conjugated through the widely used EDC–NHS method, which links the carboxyl group of MDA to the amine group of TBO. Subsequently, different amounts of MDA–TBO are mixed with the same amount of Au@4-ATP@Ag solution, and Raman measurements are carried out. The thiol group of the MDA–TBO conjugate would readily bind to the Au@4-ATP@Ag nanoparticle surface, bringing the linked TBO close to the nanoparticles. Because of its low concentration (0.1 mM), TBO in the Au@4ATP@Ag@MDA–TBO complex was not clearly shown in the extinction spectra, except a small bump at 630 nm (Fig. 2). Results of the Raman measurements are shown in Fig. 3. In the Raman spectra of pure TBO solid and Au@4-ATP@Ag under 785 nm excitation, as shown in Fig. S1a, two distinct peaks, 1585 and 1620 cm 1, are chosen to be associated with 4-ATP and TBO, respectively. SERS spectra of a series of Au@4-ATP@Ag@MDA–TBO with same amount of Au@4-ATP@Ag nanoparticles but different concentrations of TBO are shown in Fig. 3a. It is observed that, as the TBO concentration increases, the intensity of the 1620 cm 1 peak also increases. The 1620 cm 1 peak intensity of TBO is plotted against TBO concentration in Fig. 3b. As a comparison, the ratio of the peak intensity at 1620 and 1585 cm 1,ITBO/I4-ATP, is also plotted against [TBO] and shown in Fig. 3b. The improvement in the linear fitting illustrates that the use of internal reference improves the accuracy of the quantitative SERS measurements. The very good linear relationship between ITBO/I4-ATP and [TBO] allows to quantify the TBO concentration from 0.1 to 1 mM.
3.2. SERS quantification of TBO We first demonstrate the detection of Toluidine Blue O (TBO), which is brought close to the Au@4-ATP@Ag nanoparticle surface through a linker molecule, 12-mercaptododecanoic acid (MDA).
Fig. 1. Schematic illustration of quantitative SERS measurement assisted by Au-core/Ag-shell nanoparticles with embedded internal reference.
Fig. 2. UV–vis spectra of AuNPs, Au@4-ATP, Au@4-ATP@Ag and Au@4ATP@Ag@MDA–TBO (top). TEM image of Au@4-ATP@Ag@MDA–TBO (bottom).
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intensity at 700 and 1075 cm 1, Imelamine/I4-ATP, and the melamine concentration is observed. The good linear relationship between Imelamine/I4-ATP and [melamine] allows the quantification of melamine concentration in milk down to 5 mM (Fig. 4b). Comparison with the plot of the 700 cm 1 peak intensity against melamine concentration (Fig. 4b) confirms the advantage of the internal reference method for quantitative measurements. The detection of melamine in milk is less sensitive than that of TBO, probably because the adsorption efficiency of melamine in milk onto the Au@4-ATP@Ag nanoparticle surface is lower than that of TBO through MDA. Nevertheless, it demonstrates the generality of this detection scheme for analytes without the assistance of linker molecules. Some features of this strategy should be noted. The use of an internal reference embedded in the Au-core/Ag-shell nanoparticles may be versatile for the detection of multiple analytes, as in the case of this study. Besides, there are a large number of molecules that can be used as the internal reference, making it easy to find spectral fingerprints not overlapping with the target analytes. The internal reference is embedded between Au core and Ag shell while the target analyte is outside the Ag shell. Thus there is no competition between the two for the nanoparticle surface. The detection limit and the dynamic range of this scheme are associated with the size and concentration of the Au-core/Ag-shell nanoparticles. They can be improved by adjusting these parameters. The nanoparticle surface can also be functionalized with
Fig. 3. (a) Raman spectra of Au@4-ATP@Ag mixed with different concentrations of MDA–TBO: (A) 0 mM; (B) 0.1 mM; (C) 0.2 mM; (D) 0.3 mM; (E) 0.5 mM; (F) 0.6 mM; (G) 0.9 mM; (H) 1.0 mM. (b) Ratio of peak areas of TBO (1620 cm 1) and 4-ATP (1585 cm 1) vs. TBO concentration (red); peak area of TBO (1620 cm 1) vs. TBO concentration (blue). Error bars were obtained from five measurements at each concentration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. SERS quantification of melamine in milk We further apply this strategy to detect analytes without the assistance of the linker molecules, using melamine as a target molecule. Melamine (2,4,6-triamino-1,3,5-triazine) is a nitrogen-rich chemical commonly used to produce kitchenware, commercial filters, flame retardants, and other products [22]. The illegal adulteration with melamine in dairy products could cause serious kidney problems. The global food safety alarm in 2008 involving milk and infant formula indicated that milk products contaminated with melamine could lead to kidney disease and even mortality in infants [23,24]. Therefore, it is important to develop simple and reliable methods for low-cost, rapid and accurate determination of melamine. Different amounts of melamine in milk are mixed with the same amount of Au@4-ATP@Ag solution, and Raman measurements are taken. Based on the Raman spectra of pure melamine solid and Au@4-ATP@Ag under 785 nm excitation (Fig. S1b), two distinct peaks, 1075 and 700 cm 1, are chosen to be associated with 4-ATP and melamine, respectively. The melamine peak at 700 cm 1 has a slight red-shift compared with that of the solid melamine, likely due to the effect of core–shell nanoparticles on the triazine ring of melamine [25]. SERS spectra of a series of Au@4ATP@Ag + melamine with the same amount of Au@4-ATP@Ag nanoparticles but different concentrations of melamine are shown in Fig. 4a. Again, a strong correlation between the ratio of the peak
Fig. 4. (a) Raman spectra of Au@4-ATP@Ag mixed with different concentrations of melamine in milk: (A) 0 mM; (B) 5 mM; (C) 10 mM; (D) 25 mM; (E) 50 mM; (F) 75 mM; (G) 100 mM. (b) Ratio of peak areas of melamine (700 cm 1) and 4-ATP (1075 cm 1) vs. melamine concentration (red); peak area of melamine (700 cm 1) vs. melamine concentration (blue). Error bars were obtained from five measurements at each concentration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Y. Zhou et al. / Analytica Chimica Acta 874 (2015) 49–53
capturing elements to target specific analytes of interest to improve the sensitivity. 4. Conclusions In summary, we introduce a new strategy to quantitatively measure the SERS signals of analytes based on Au-core/Ag-shell nanoparticles with embedded internal reference. Successful detections of two analytes, TBO and melamine, are demonstrated. The adoption of a linker molecule in the detection of TBO leads to the higher sensitivity, while the detection of melamine without any linker molecules illustrates the versatility of this scheme for a wide variety of analytes. For analytes in a specific setting, the experimental conditions can be optimized to further improve the detection sensitivity. It is expected that this strategy will have great implication to quantitative SERS measurements of a broad range of analytes. Acknowledgement This work is partially supported by the US National Science Foundation (CBET-1065633). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2015.03.016. References [1] D.A. Stuart, J.M. Yuen, N. Shah, O. Lyandres, C.R. Yonzon, M.R. Glucksberg, J.T. Walsh, R.P. Van Duyne, In vivo glucose measurement by surface-enhanced Raman spectroscopy, Anal. Chem. 78 (2006) 7211–7215. [2] S. Schlücker, Surface-enhanced Raman spectroscopy: concepts and chemical applications, Angew. Chem. Int. Ed. 53 (2014) 4756–4795. [3] J.F. Betz, W.W. Yu, Y. Cheng, I.M. White, G.W. Rubloff, Simple SERS substrates: powerful, portable, and full of potential, Phys. Chem. Chem. Phys. 16 (2014) 2224–2239. [4] K.C. Bantz, A.F. Meyer, N.J. Wittenberg, H. Im, O. Kurtuluş, S.H. Lee, N.C. Lindquist, S.H. Oh, C.L. Haynes, Recent progress in SERS biosensing, Phys. Chem. Chem. Phys. 13 (2011) 11551–11567. [5] Y.S. Huh, A.J. Chung, D. Erickson, Surface enhanced Raman spectroscopy and its application to molecular and cellular analysis, Microfluid. Nanofluid. 6 (2009) 285–297.
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[6] M.B. Wabuyele, T. Vo-Dinh, Detection of human immunodeficiency virus type 1 DNA sequence using plasmonics nanoprobes, Anal. Chem. 77 (2005) 7810–7815. [7] C.J. McHugh, R. Keir, D. Graham, W.E. Smith, Selective functionalisation of TNT for sensitive detection by SERRS, Chem. Commun. (2002) 580–581. [8] Y.C. Cao, R. Jin, C.A. Mirkin, Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection, Science 297 (2002) 1536–1540. [9] Y.C. Cao, R. Jin, J.-M. Nam, C.S. Thaxton, C.A. Mirkin, Raman dye-labeled nanoparticle probes for proteins, J. Am. Chem. Soc. 125 (2003) 14676–14677. [10] S. Chan, S. Kwon, T.W. Koo, L.P. Lee, A.A. Berlin, Surface-enhanced Raman scattering of small molecules from silver-coated silicon nanopores, Adv. Mater. 15 (2003) 1595–1598. [11] W.E. Doering, S. Nie, Spectroscopic tags using dye-embedded nanoparticles and surface-enhanced Raman scattering, Anal. Chem. 75 (2003) 6171–6176. [12] D.K. Lim, K.S. Jeon, H.M. Kim, J.M. Nam, Y.D. Suh, Nanogap-engineerable Raman-active nanodumbbells for single-molecule detection, Nat. Mater. 9 (2010) 60–67. [13] P. Pinkhasova, B. Puccio, T. Chou, S. Sukhishvili, H. Du, Noble metal nanostructure both as a SERS nanotag and an analyte probe, Chem. Commun. 48 (2012) 9750–9752. [14] W. Li, Y. Guo, P. Zhang, A general strategy to prepare TiO2-core gold-shell nanoparticles as SERS-tags, J. Phys. Chem. C 114 (2010) 7263–7268. [15] W.B. Lacy, L.G. Olson, J.M. Harris, Quantitative SERS measurements on dielectric-overcoated silver-island films by solution-deposition control of surface concentrations, Anal. Chem. 71 (1997) 2564–2570. [16] A. Campion, P. Kambhampati, Surface-enhanced Raman scattering, Chem. Soc. Rev. 27 (1998) 241–250. [17] A. Loren, J. Engelbrektsson, C. Eliasson, M. Josefson, J. Abrahamsson, M. Johansson, K. Abrahamsson, Internal standard in surface-enhanced Raman spectroscopy, Anal. Chem. 76 (2004) 7391–7395. [18] D.M. Zhang, Y. Xie, S.K. Deb, V.J. Davisson, D. Ben-Amotz, Isotope edited internal standard method for quantitative surface-enhanced Raman spectroscopy, Anal. Chem. 77 (2005) 3563–3569. [19] S.K. Deb, B. Davis, G.M. Knudsen, R. Gudihal, D. Ben-Amotz, V.J. Davisson, Detection and relative quantification of proteins by surface enhanced Raman using isotopic labels, J. Am. Chem. Soc. 130 (2008) 9624–9625. [20] G. Frens, Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions, Nat. Phys. Sci. 241 (1973) 20–22. [21] Y. Zhou, C. Lee, J. Zhang, P. Zhang, Engineering versatile SERS-active nanoparticles by embedding reporters between Au-core/Ag-shell through layer-by-layer deposited polyelectrolytes, J. Mater. Chem. C 1 (2013) 3695–3699. [22] World Health Organization. Melamine and Cyanuric Acid: Toxicity, Preliminary Risk Assessment and Guidance on Levels in Food, October 3, 2008. [23] J.R. Ingelfinger, N. Engl, Melamine and the global implications of food contamination, J. Med. 359 (2008) 2745–2748. [24] H. Xin, R. Stone, Chinese probe unmasks high-tech adulteration with melamine, Science 322 (2008) 1310–1311. [25] P. Ma, F. Liang, Y. Sun, Y. Jin, Y. Chen, X. Wang, H. Zhang, D. Gao, D. Song, Rapid determination of melamine in milk and milk powder by surface-enhanced Raman spectroscopy and using cyclodextrin-decorated silver nanoparticles, Microchim. Acta 180 (2013) 1173–1180.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Quantitative surface-enhanced Raman measurements with embedded internal reference Yan Zhou, Rui Ding, Padmanabh Joshi, Peng Zhang * Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, United States
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A new methodology to perform quantitative SERS measurements of analytes using internal reference. SERS-based quantification of Toluidine Blue O as a proof-of-concept study. SERS-based quantification of melamine in milk.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 August 2014 Received in revised form 21 November 2014 Accepted 9 March 2015 Available online 11 March 2015
Analytical applications of SERS are often more associated with qualitative than quantitative analysis, because of the difficulty in obtaining quantitative SERS results. In this paper we introduce a new strategy to quantitatively measure the SERS signals of analytes based on Au-core/Ag-shell nanoparticles with embedded 4-aminothiophenol as the internal reference. Successful detections of two analytes, Toluidine Blue O in aqueous solution (detection limit of 0.1 mM) and melamine in milk (detection limit of 5 mM), are demonstrated. The improvement in the linear fitting illustrates that the use of internal reference significantly improves the accuracy of the quantitative SERS measurements. The successful detection of melamine in milk illustrates the versatility of this detection scheme for a wide variety of analytes. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Quantitative surface-enhanced Raman measurement Internal reference Aminothiophenol Melamine Core–shell nanostructure
1. Introduction
* Corresponding author. Tel.: +1 513 556 9222. E-mail address: [email protected] (P. Zhang). http://dx.doi.org/10.1016/j.aca.2015.03.016 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.
Surface-enhanced Raman spectroscopy (SERS) has proved to be a useful tool for diagnostic applications, and detection of explosives and bioanalytes [1–9]. Raman scattering of molecules, despite of being an inefficient process with very small crosssections, can be greatly enhanced by orders of magnitude if the
50
Y. Zhou et al. / Analytica Chimica Acta 874 (2015) 49–53
molecules reside in the vicinity of some metal nanostructures, especially those of silver and gold [10,11]. The highly enhanced Raman signals have allowed SERS to be used as a tool in singlemolecule spectroscopy [12]. Due to the inherently narrow and fingerprint-specific spectra, SERS offers a greater degree of multiplexing capability, as compared to the more widely used fluorescence-based detection schemes [13,14]. However, analytical applications of SERS are often more associated with qualitative than quantitative analysis, because of the difficulty in obtaining quantitative SERS results. Quantitative SERS measurements are usually jeopardized by several factors, such as poor reproducibility of the SERS substrates, and variation in the adsorption of analyte to the substrate [15,16]. There have been efforts to address issues associated with the quantification aspect of SERS. For instance, a method was proposed to improve the accuracy of SERS measurements by using the SERS signal generated from a self-assembled monolayer (SAM) as the internal reference [17]. SAMs prevent chemisorption of the analyte onto the SERS-active surface and thus improve the reproducibility. Yet the detection limit and the dynamic range have been greatly compromised because of the distance between the analyte and SERS-active surface created by the SAM coating. A different group has introduced a so-called isotope edited internal standard method to improve the accuracy of quantitative SERS measurement [18,19]. An isotope-edited version of the analyte was used as the internal reference to minimize the difference in enhancement factors of the analyte and the reference. In practice, this approach is hindered by some intrinsic limitations. For instance, an isotope-edited counterpart is required for any given analyte of interest. Additionally, the SERS spectral features of the analyte and its isotope-edited internal standard have to be sufficiently different to facilitate independent measurement of their SERS intensities in a mixture. Since most molecules have very similar SERS spectral features with their isotope-edited counterparts, the types of intended analytes are limited. Competition between the analyte and its isotope-edited counterpart may cause extra uncertainty as both are directly attached to the same metal surface. Herein we report a new strategy for quantitative SERS measurements through an internal reference embedded inside the Au-core/Ag-shell nanoparticles, which serve as SERS substrates for analytes in the proximity. The scheme provides great flexibility in the choice of the internal references, which should be different from the analytes, and allows quantitative SERS measurements of the analytes in the solution. Two different types of analytes, Toluidine Blue O (TBO) and melamine, are studied in this paper. The TBO sensing is shown as a proof-of-concept study, while the melamine sensing in milk illustrates the practicality of this strategy in real applications.
2.2. Synthesis of citrate stabilized Au nanoparticles Gold nanoparticles of 20 nm were synthesized according to the procedures in the literature [20]. Briefly, in a 100-mL glass flask, 50 mL of a 0.01% aqueous HAuCl4 solution was heated to boil under magnetic stirring. Upon boiling, 1 mL of 1% sodium citrate was rapidly injected. Within seconds, the pale yellow solution turned into deep purple and quickly progressed to red. The colloid was kept boiling for 15 min to ensure complete reduction, before left to cool to room temperature. Excessive ions were removed from the solution by centrifugation, and the AuNPs were redispersed in 50 mL of DI water. The AuNPs solution was kept at 4 C until later use. 2.3. Synthesis of Au@ATP@Ag nanoparticles 10 mL of 4-ATP (0.1 mM in EtOH) was added to 10 mL as-synthesized citrate-stabilized AuNPs solution under vortex. The solution was incubated overnight at room temperature. Deposition of silver shell onto the 4-ATP-conjugated gold nanoparticles was similar to the method in the literature with minor modification [21]. Hydroquinone (300 mL, 10 mM in water) and AgNO3 (300 mL, 10 mM in water) were added in sequence to 1 mL Au@4-ATP solution under vigorous stirring. The mixture was incubated at room temperature for 12 h to allow for the completion of the reaction. The solution was then centrifuged for 10 min at 14,000 rpm to collect the precipitate, which was re-dispersed in 10 mL DI water until later use. 2.4. Synthesis of MDA–TBO conjugates In a typical reaction, MDA (23 mg, 0.01 mM) was dissolved in a mixture of 1 mL ethanol and 9 mL of 50 mM MES buffer (pH 6.5). Then, 20 mg EDC and 30 mg NHS was added in sequence to the solution. The activation reaction was allowed to proceed at room temperature for 20 min. TBO (31 mg, 0.01 mM) was then added to the solution under stirring. After stirring for 24 h, the final solution was diluted to 10 mM for later use. 2.5. Characterization UV–vis extinction spectra were recorded on a USB 4000 spectrophotometer (Ocean Optics, USA) using a 1-cm path length quartz cell at room temperature. TEM measurements were performed on a Biotwin 12 transmission electron microscope (FEI). TEM samples were prepared by depositing a drop of suspension containing nanoparticles on a formvar-covered carbon-coated copper grid (Electron Microscopy Sciences, PA) and letting it dried at room temperature.
2. Materials and methods
2.6. Raman measurement
2.1. Chemicals and materials
All samples for Raman measurement were prepared by adding a known amount of an analyte solution into Au@4-ATP@Ag solution. For TBO samples, MDA–TBO (10 mL, 10 mM) was added into 990 mL as-synthesized Au@4-ATP@Ag solution under magnetic stirring. By changing the concentration of MDA–TBO from 10 mM to 20, 30, 50, 60, 90 and 100 mM, the other six solutions were obtained for later use. Melamine was first dissolved in DI water to prepare a 10 mM stock solution. The melamine solution was next spiked into the milk purchased from a local grocery store, to obtain milk solutions containing 0.05, 0.1, 0.25, 0.5, 0.75 and 1 mM melamine, respectively. Then, samples with melamine concentration of 5, 10, 25, 50, 75, and 100 mM, respectively, were obtained by adding 100 mL of the melamine-containing milk solutions into 900 mL of as-synthesized Au@4-ATP@Ag solution. Raman measurements
4-Aminothiophenol (4-ATP), Toluidine Blue O (TBO), melamine, hydrogen tetrachloroaurate, 12-mercaptododecanoic acid (MDA), 2-(N-morpholino)ethanesulfonic acid (MES), sodium citrate, hydroquinone, sodium cyanide, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were obtained from Sigma–Aldrich. Milk was purchased in a local grocery store (Meijer). All chemicals, unless specified, were of reagent grade. Reagents and solvents were obtained commercially and used without further purification. All glassware were cleaned with freshly prepared aqua regia and rinsed thoroughly with DI water prior to use. All solutions were prepared using deionized (DI) water (18 MV-cm).
Y. Zhou et al. / Analytica Chimica Acta 874 (2015) 49–53
were done on a Renishaw inVia Raman microscope system. The aforementioned samples were dispersed thoroughly by sonication. A drop of the dispersion was transferred into a small quartz tube of 1 mm I.D. The tube was placed on the stage of the microscope for Raman measurements. The microscope objective has 10 magnification and 0.25 NA. Laser intensity at the samples was 11 mW using the 785 nm line of a diode laser for all measurements. Exposure time was 10 s for the TBO measurements and 20 s for melamine measurements. Between different Raman sessions, the 520.7 cm 1 peak of a silicon wafer was used to calibrate the spectrograph. Fig. S1 shows the Raman spectra of Au@4-ATP@Ag, solid TBO and solid melamine (Supporting information). 3. Results and discussion 3.1. Synthesis and characterization The design of the Au-core/Ag-shell nanoparticle with embedded internal reference is illustrated in Fig. 1. The method to synthesize internal reference-embedded Au-core/Ag-shell nanoparticles is similar to what was reported previously with minor modifications [21]. In brief, gold nanoparticles are first synthesized through thermal reduction of HAuCl4 by citrate according to the literature [20], and washed thoroughly before use. Probe molecules serving as internal reference are adsorbed to the gold nanoparticle surface, on top of which a silver layer is subsequently deposited. The probe molecule used in this work is 4-aminothiophenol (4-ATP). The formation of the Au-core/Ag-shell nanoparticles is confirmed by both UV–vis extinction spectroscopy and transmission electron microscopy (TEM), as shown in Fig. 2. The extinction spectrum of gold nanoparticles with 4-ATP attached (Au@4-ATP) includes the typical plasmon band of Au nanoparticles (520 nm). The spectrum of Au@4-ATP@Ag contains a broad shoulder band at 400 nm and a slightly blue-shifted Au plasmon band, both indicating the formation of the Ag-shell on the Au@4-ATP nanoparticles. The distinct core–shell structure can be observed in the TEM image (Fig. 2, Fig. S2). The Au@4-ATP@Ag nanoparticles are well dispersed in water and stable for weeks.
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TBO and MDA are conjugated through the widely used EDC–NHS method, which links the carboxyl group of MDA to the amine group of TBO. Subsequently, different amounts of MDA–TBO are mixed with the same amount of Au@4-ATP@Ag solution, and Raman measurements are carried out. The thiol group of the MDA–TBO conjugate would readily bind to the Au@4-ATP@Ag nanoparticle surface, bringing the linked TBO close to the nanoparticles. Because of its low concentration (0.1 mM), TBO in the Au@4ATP@Ag@MDA–TBO complex was not clearly shown in the extinction spectra, except a small bump at 630 nm (Fig. 2). Results of the Raman measurements are shown in Fig. 3. In the Raman spectra of pure TBO solid and Au@4-ATP@Ag under 785 nm excitation, as shown in Fig. S1a, two distinct peaks, 1585 and 1620 cm 1, are chosen to be associated with 4-ATP and TBO, respectively. SERS spectra of a series of Au@4-ATP@Ag@MDA–TBO with same amount of Au@4-ATP@Ag nanoparticles but different concentrations of TBO are shown in Fig. 3a. It is observed that, as the TBO concentration increases, the intensity of the 1620 cm 1 peak also increases. The 1620 cm 1 peak intensity of TBO is plotted against TBO concentration in Fig. 3b. As a comparison, the ratio of the peak intensity at 1620 and 1585 cm 1,ITBO/I4-ATP, is also plotted against [TBO] and shown in Fig. 3b. The improvement in the linear fitting illustrates that the use of internal reference improves the accuracy of the quantitative SERS measurements. The very good linear relationship between ITBO/I4-ATP and [TBO] allows to quantify the TBO concentration from 0.1 to 1 mM.
3.2. SERS quantification of TBO We first demonstrate the detection of Toluidine Blue O (TBO), which is brought close to the Au@4-ATP@Ag nanoparticle surface through a linker molecule, 12-mercaptododecanoic acid (MDA).
Fig. 1. Schematic illustration of quantitative SERS measurement assisted by Au-core/Ag-shell nanoparticles with embedded internal reference.
Fig. 2. UV–vis spectra of AuNPs, Au@4-ATP, Au@4-ATP@Ag and Au@4ATP@Ag@MDA–TBO (top). TEM image of Au@4-ATP@Ag@MDA–TBO (bottom).
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intensity at 700 and 1075 cm 1, Imelamine/I4-ATP, and the melamine concentration is observed. The good linear relationship between Imelamine/I4-ATP and [melamine] allows the quantification of melamine concentration in milk down to 5 mM (Fig. 4b). Comparison with the plot of the 700 cm 1 peak intensity against melamine concentration (Fig. 4b) confirms the advantage of the internal reference method for quantitative measurements. The detection of melamine in milk is less sensitive than that of TBO, probably because the adsorption efficiency of melamine in milk onto the Au@4-ATP@Ag nanoparticle surface is lower than that of TBO through MDA. Nevertheless, it demonstrates the generality of this detection scheme for analytes without the assistance of linker molecules. Some features of this strategy should be noted. The use of an internal reference embedded in the Au-core/Ag-shell nanoparticles may be versatile for the detection of multiple analytes, as in the case of this study. Besides, there are a large number of molecules that can be used as the internal reference, making it easy to find spectral fingerprints not overlapping with the target analytes. The internal reference is embedded between Au core and Ag shell while the target analyte is outside the Ag shell. Thus there is no competition between the two for the nanoparticle surface. The detection limit and the dynamic range of this scheme are associated with the size and concentration of the Au-core/Ag-shell nanoparticles. They can be improved by adjusting these parameters. The nanoparticle surface can also be functionalized with
Fig. 3. (a) Raman spectra of Au@4-ATP@Ag mixed with different concentrations of MDA–TBO: (A) 0 mM; (B) 0.1 mM; (C) 0.2 mM; (D) 0.3 mM; (E) 0.5 mM; (F) 0.6 mM; (G) 0.9 mM; (H) 1.0 mM. (b) Ratio of peak areas of TBO (1620 cm 1) and 4-ATP (1585 cm 1) vs. TBO concentration (red); peak area of TBO (1620 cm 1) vs. TBO concentration (blue). Error bars were obtained from five measurements at each concentration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. SERS quantification of melamine in milk We further apply this strategy to detect analytes without the assistance of the linker molecules, using melamine as a target molecule. Melamine (2,4,6-triamino-1,3,5-triazine) is a nitrogen-rich chemical commonly used to produce kitchenware, commercial filters, flame retardants, and other products [22]. The illegal adulteration with melamine in dairy products could cause serious kidney problems. The global food safety alarm in 2008 involving milk and infant formula indicated that milk products contaminated with melamine could lead to kidney disease and even mortality in infants [23,24]. Therefore, it is important to develop simple and reliable methods for low-cost, rapid and accurate determination of melamine. Different amounts of melamine in milk are mixed with the same amount of Au@4-ATP@Ag solution, and Raman measurements are taken. Based on the Raman spectra of pure melamine solid and Au@4-ATP@Ag under 785 nm excitation (Fig. S1b), two distinct peaks, 1075 and 700 cm 1, are chosen to be associated with 4-ATP and melamine, respectively. The melamine peak at 700 cm 1 has a slight red-shift compared with that of the solid melamine, likely due to the effect of core–shell nanoparticles on the triazine ring of melamine [25]. SERS spectra of a series of Au@4ATP@Ag + melamine with the same amount of Au@4-ATP@Ag nanoparticles but different concentrations of melamine are shown in Fig. 4a. Again, a strong correlation between the ratio of the peak
Fig. 4. (a) Raman spectra of Au@4-ATP@Ag mixed with different concentrations of melamine in milk: (A) 0 mM; (B) 5 mM; (C) 10 mM; (D) 25 mM; (E) 50 mM; (F) 75 mM; (G) 100 mM. (b) Ratio of peak areas of melamine (700 cm 1) and 4-ATP (1075 cm 1) vs. melamine concentration (red); peak area of melamine (700 cm 1) vs. melamine concentration (blue). Error bars were obtained from five measurements at each concentration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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capturing elements to target specific analytes of interest to improve the sensitivity. 4. Conclusions In summary, we introduce a new strategy to quantitatively measure the SERS signals of analytes based on Au-core/Ag-shell nanoparticles with embedded internal reference. Successful detections of two analytes, TBO and melamine, are demonstrated. The adoption of a linker molecule in the detection of TBO leads to the higher sensitivity, while the detection of melamine without any linker molecules illustrates the versatility of this scheme for a wide variety of analytes. For analytes in a specific setting, the experimental conditions can be optimized to further improve the detection sensitivity. It is expected that this strategy will have great implication to quantitative SERS measurements of a broad range of analytes. Acknowledgement This work is partially supported by the US National Science Foundation (CBET-1065633). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2015.03.016. References [1] D.A. Stuart, J.M. Yuen, N. Shah, O. Lyandres, C.R. Yonzon, M.R. Glucksberg, J.T. Walsh, R.P. Van Duyne, In vivo glucose measurement by surface-enhanced Raman spectroscopy, Anal. Chem. 78 (2006) 7211–7215. [2] S. Schlücker, Surface-enhanced Raman spectroscopy: concepts and chemical applications, Angew. Chem. Int. Ed. 53 (2014) 4756–4795. [3] J.F. Betz, W.W. Yu, Y. Cheng, I.M. White, G.W. Rubloff, Simple SERS substrates: powerful, portable, and full of potential, Phys. Chem. Chem. Phys. 16 (2014) 2224–2239. [4] K.C. Bantz, A.F. Meyer, N.J. Wittenberg, H. Im, O. Kurtuluş, S.H. Lee, N.C. Lindquist, S.H. Oh, C.L. Haynes, Recent progress in SERS biosensing, Phys. Chem. Chem. Phys. 13 (2011) 11551–11567. [5] Y.S. Huh, A.J. Chung, D. Erickson, Surface enhanced Raman spectroscopy and its application to molecular and cellular analysis, Microfluid. Nanofluid. 6 (2009) 285–297.
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