Article pubs.acs.org/ac
Self-Assembly of Au Nanoparticles on PMMA Template as Flexible, Transparent, and Highly Active SERS Substrates Lu-Bin Zhong,† Jun Yin,† Yu-Ming Zheng,*,† Qing Liu,† Xiao-Xia Cheng,† and Fang-Hong Luo‡ †
Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Road, Xiamen 361021, P. R. China Cancer Research Center, Medical College, Xiamen University, 422 South Siming Road, Xiamen 361005, P. R. China
‡
S Supporting Information *
ABSTRACT: We report a simple and rapid method for fabricating a surface-enhanced Raman scattering (SERS) substrate, which offers good flexibility, excellent optical transparency, and high SERS activity. Specifically, the SERS substrate (AuNPs/PMMA film) was obtained through self-assembly of gold nanoparticles (AuNPs) on newborn poly(methyl methacrylate) (PMMA) template. The UV−vis spectroscopy analysis and scanning electron microscopy observation revealed that the gold nanoparticles were closely assembled on the flexible and transparent PMMA template. The fabricated AuNPs/PMMA film SERS substrate allowed detection of model molecule, malachite green isothiocyanate, at a concentration as low as 0.1 nM, and exhibited good reproducibility in the SERS measurement. The Raman enhancement factor (EF) of the AuNPs/PMMA film was found to be as high as (2.4 ± 0.3) × 107. In addition, measure of residual malachite green on fish surface was carried out, and the result indicated that the AuNPs/PMMA film had great potential in the in situ ultrasensitive detection of analyte on irregular objects.
S
detached from the paper because of the weak physical adsorption. To firmly fix the nanoparticles on the templates, usually chemical modifications of nanoparticles or templates were required, which was rather tedious.9,10 To save the process of chemical modification, Hasell et al. took advantage of the physical constraint of the polymer template to fix nanoparticles.11 Unfortunately, this method requires 24 h heating under high pressure to dissolve the precursor complex and infuse it into the polymer. Recently, Yu et al. presented a new class of SERS substrates fabricated via electrospinning technique.12,13 However, because the nanoparticles were incorporated inside opaque nanofibers, which resulted in the reduction of in situ detection sensitivity. Hence, it is an urgent need to develop a simple, rapid and cost-efficient way for fabrication of flexible SERS substrate. Herein, we present a novel fabrication strategy to produce highly active SERS substrates with good flexibility and optical transparency. More importantly, the SERS substrates can be easily and quickly prepared without any expensive instrument. The authors demonstrate the method for constructing flexible SERS substrates through self-assembly of Au nanoparticles (AuNPs) in newborn poly(methyl methacrylate) (PMMA) template, which is denoted as AuNPs/PMMA film. The preparation procedure is shown in Scheme 1.
urface-enhanced Raman scattering (SERS) has been recognized as a powerful molecular spectroscopic technique, which allows nondestructive chemical or biochemical analysis with ultrasensitive detection.1,2 Two mechanisms have been widely accepted to account for the SERS effect, chemical enhancement, and electromagnetic enhancement. Compared to chemical enhancement, the strong electromagnetic field enhancement near metallic nanoparticles is mainly responsible for the occurrence of SERS.3,4 For this reason, research interests are turning toward developing advanced metal nanoparticle-based SERS substrates, which are capable of generating high-quality Raman scattering signal from interested analytes.5,6 However, most of works were devoted to fabricate SERS substrates with hard templates (such as glass, quartz, or silicon). Because of lack of flexibility, the target analytes on irregular shaped objects need to be extracted with suitable solvent before SERS analyzing, which limits its application to in situ detection of analytes on planar objects only.7 Therefore, there is a strong need and has an increasing interest in the development of flexible, transparent and highly active SERS substrates, which are capable of providing in situ ultrasensitive SERS analysis of analytes on objects with diverse morphologies.7 Although there are some reports on the preparation of flexible SERS substrates, the methods have some drawbacks. For example, Lee et al. reported the fabrication of flexible SERS substrates by dipping a paper into the nanoparticles solution.8 The method was simple, but it required a rather concentrated nanoparticle solution, and the nanoparticles were easily © XXXX American Chemical Society
Received: December 28, 2013 Accepted: May 29, 2014
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dx.doi.org/10.1021/ac404224f | Anal. Chem. XXXX, XXX, XXX−XXX
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Poly(methyl methacrylate) (PMMA, MW = 1.2 × 105) was purchased from Sigma-Aldrich. Malachite green isothiocyanate (MGITC) was purchased from Invitrogen Corporation, Carlsbad, CA. Fish were purchased from local supermarket. Synthesis of AuNPs. AuNPs were prepared according to the method reported by Carpay and Cense with minor modification.20 In a typical synthesis, 1 mL of sodium citrate solution (38.8 mM) was quickly added to 99 mL of aqueous HAuCl4 solution (0.243 mM) that was stirred in boiling condition, and the mixture was boiled for 5 min to give a winered solution. Another 1 mL of sodium citrate solution (38.8 mM) and 1 mL of HAuCl4 solution (0.243 mM) were then successively added to the mixture every 20 s, and this step was repeated by 3 times. After that, heating was stopped, and the reaction mixture was allowed to cool down to room temperature. The mean size of the prepared AuNPs was 55 ± 5 nm. Fabrication of AuNPs/PMMA SERS Substrate. First, 15 mL of freshly prepared AuNPs suspension was transferred to a beaker, followed by addition of 7 mL of toluene with different concentrations of PMMA (0−3 mg/cm2). Because of the immiscibility of water and toluene, a clear water/toluene interface was formed. After that, 8 mL of ethanol was injected into the AuNPs suspension using a mechanical syringe pump (LSPO4-1A, Baoding Longer Precision Pump Co., Ltd.) with a feeding rate of 10 mL/h. With the addition of ethanol, the AuNPs rose up to the water/toluene interface and selfassembled into orderly AuNPs layers. A thin PMMA template was simultaneously formed on the top of the aqueous solution because of the evaporation of toluene. The self-assembled AuNPs layer was then fixed on the newborn PMMA template. When the toluene fully evaporated, a polyethylene (PE) film was used as a carrier to retrieve the prepared AuNPs/PMMA film from the top of the aqueous solution surface. Finally, the PE film carried AuNPs/PMMA film was dried at 60 °C in a vacuum oven, and used as SERS substrate in the subsequent experiments. The PE film was found to be fastened tightly to the AuNPs/PMMA film, and was not detached from the AuNPs/PMMA film in the SERS detection process. The preparation process did not involve tedious procedures and expensive equipment, and the whole process can be completed within a few hours. Preparation of SERS Samples. To determine SERS enhancement factor, SERS sample 1 was prepared as follows: The prepared AuNPs/PMMA film was immersed in 10−5 M MGITC ethanol solution for 20 min. The AuNPs/PMMA film was then washed with ethanol to remove the unbound MGITC molecules and dried at room temperature to evaporate all of the ethanol. To evaluate the applicability of the prepared AuNPs/PMMA film as a SERS substrate for in situ analyte detection, SERS sample 2 was prepared. Fish were immersed in solutions with different MGITC concentrations for 10 min. The AuNPs/ PMMA film was wetted with water and the side exposing AuNPs was attached to fish surface for direct SERS measurement. Characterization. Optical characterization was carried out by a UV−vis spectrophotometer (UV-759, China). SEM images were obtained with a field-emission scanning electron microscope (Hitachi S-4800, Japan). SERS spectra were recorded using a confocal microscope Raman spectrometer (LabRAM Aramis, France). A 785 nm semiconductor diode laser was used for Raman excitation, which was focused onto a
Scheme 1. Schematic Representation of Preparation of AuNPs/PMMA SERS Substratea
a
Typical photographs of the prepared SERS substrate are demonstrated in the top right corner.
First, AuNPs were well dispersed in aqueous solution, and then PMMA toluene solution was added gently to the top of the AuNPs solution. After that, ethanol was added dropwise to the AuNPs solution and induced the AuNPs to rise slowly to the water/toluene interface. With the addition of ethanol, more and more AuNPs rose up and self-assembled into orderly layer with innumerable “hot spots”.14,15 In the meantime, the PMMA molecules built up at the water/toluene interface to form a thin PMMA template because of the evaporation of toluene. The newborn thin PMMA film served as a template to fix the selfassembled AuNPs layer by the physical constraint. One side of the AuNPs layer was tightly wrapped by the PMMA template, the opposite side was unattached and exposed to the aqueous solution. It is expected that the exposed AuNPs layer can directly contact with analytes and enhance the characteristic Raman signal maximally. After the complete evaporation of toluene, a polyethylene (PE) film was used as a carrier to retrieve the thin AuNPs/ PMMA film from the top of aqueous solution, as the AuNPs/ PMMA film is too thin to be taken out. The whole process can be completed in a short time without any expensive equipment. PMMA was chosen as an ideal template polymer for fixing the self-assembled AuNPs layers because of its excellent optical transparency. Light can pass through PMMA template and reach AuNPs layer to activate the plasmon resonance, which gives rise to the enormous SERS enhancement. Moreover, because of PMMA has a low Raman cross-section, the AuNPs/ PMMA film is expected to exhibit clear SERS signals.16,17 As shown in the photographs of Scheme 1, the AuNPs/PMMA film can be bended sharply, implying its good flexibility. The side of AuNPs layer of AuNPs/PMMA film exhibits metallic luster because of electronic coupling of closely packed AuNPs.18,19 The other side of AuNPs/PMMA film (PMMA template layer) is transparent, and laser can go through it to reach the AuNPs layer, herein we define the AuNPs/PMMA film as a transparent SERS substrate.
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EXPERIMENTAL SECTION Chemical Reagents. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4 ·3H 2 O, 99.9%), sodium citrate (C 6 H 5 Na 3 O 7 , 99%), ethanol (C 2 H 6 O, 99%), toluene (C6H5CH3, 99%), and malachite green (C23H25CIN2) were purchased from Sinopharm Chemical Reagent Co., Ltd. B
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Figure 1. (a) UV−vis spectra of monodispersed AuNPs, AuNPs/PMMA film and MGITC solution. (The inset is a SEM image of the AuNPs/ PMMA film.) (b) SERS spectra of the AuNPs/PMMA film immersed in different concentration of MGITC solutions. (c) SERS spectra of MGITC collected from both sides of the AuNPs/PMMA film. Experimental conditions: the AuNPs/PMMA film containing 0.8 mg/cm2 PMMA; Raman reporter molecule is MGITC; laser wavelength =7 85 nm; laser power on the sample = 0.2 mW; integration time = 5 s.
sample with a spot size of approximately 3 μm2. The SERS measurements were performed in a confocal microscope using a 50× objective (Leica) with a numerical aperture of 0.55.
enhancement is mainly attributed to surface-enhanced Raman scattering instead of resonance enhancement.22 The Raman enhancement factor (EF) was used to evaluate the SERS activity of the prepared AuNPs/PMMA film quantitatively. The formula of EF is shown as below.23
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RESULTS AND DISCUSSION The UV−vis spectra in Figure 1a showed that the monodispersed AuNPs displayed a strong characteristic band at 550 nm, while the AuNPs/PMMA film exhibited two absorption bands. A peak at 558 nm was near to the absorption band of monodispersed AuNPs, and the other one was around 900 nm. The new absorption band can be ascribed to strong coupling between adjacent AuNPs, indicating the formation of assembled AuNPs layers on the PMMA template.21 SEM observation confirmed this hypothesis. The inset in Figure 1a showed that AuNPs were self-assembled into highly ordered layers in PMMA, and numerous “hot spots” had formed, which could significantly amplify Raman signal of analytes adsorbed on its surface (More SEM images with different magnification were provide in Supporting Information Figure S1). Although PMMA was close to AuNPs, due to its weak Raman signal, it gave a rather “clean” Raman background even after enhancement by the assembled AuNPs (see details in section 2 in the Supporting Information). To assess the detection sensitivity of the AuNPs/PMMA film, SERS spectra of AuNPs/PMMA films immersed in different concentrations of MGITC were measured. As shown in Figure 1b, the Raman characteristic bands of MGITC were clearly shown even when the MGITC concentration was reduced to 0.1 nM. Figure 1a showed the characteristic peak of MGITC UV−vis spectrum was 620 nm, which was far away from the excitation wavelength at 785 nm. Thus, Raman
EF =
ISERS/NSERS cN σhI ≈ A SERS INRS/NNRS RINRS
(1)
The obtained result showed that the EF of AuNPs/PMMA film was as high as (2.4 ± 0.3) × 107 (see details in section 3 in the Supporting Information). Besides, two other molecules, 4aminothiophenol and 4-mercaptopyridine were also used to evaluate Raman EF of the AuNPs/PMMA film under the same condition. The results showed that they had similar EF, (3.6 ± 0.5) × 107 and (9.0 ± 1.0) × 107, respectively, indicating that the AuNPs/PMMA film was able to greatly enhance the Raman signal of other components (Supporting Information Figure S3). As PMMA possesses excellent optical transparency, we presume that laser can easily pass through PMMA template and reach the assembled AuNPs layer. On this occasion, plasmon resonance of the assembled AuNPs layer would be motivated, and then the local electromagnetic field could be greatly enhanced, which markedly amplifies the Raman signal of analytes adsorbed on AuNPs layer surface.4 As shown in Figure 1c, the SERS intensities collected from both sides of the AuNPs/PMMA film were almost identical, which verified our supposition. Hence, it was expected that the SERS signal directly collected from the side of PMMA would not be sacrificed when the film was wrapped over an object surface. C
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Figure 2. (a) UV−vis spectra of the AuNPs/PMMA SERS substrate with the increment of PMMA concentration. (b) SERS spectra of the AuNPs/ PMMA SERS substrate immersed in 10−5 M MGITC with the increment of PMMA concentration. Laser wavelength = 785 nm; laser power on the sample = 0.2 mW; integration time = 5 s.
Figure 3. In situ detection of MG on fish surface. Experimental conditions: Laser wavelength = 785 nm; laser power on the sample = 2 mW; integration time = 20 s.
indicating a good homogeneity of the film (Supporting Information Figure S4d). A noteworthy fact was that PMMA content significantly affected the structure and property of the assembled AuNPs layer. If there was no PMMA in toluene, the assembled AuNPs at the water/toluene interface was unable to form complete AuNPs layers on the PE film (Supporting Information Figure S5a). The reason may be that PMMA serve as a template to fix the assembled AuNPs layers. When the assembled AuNPs layer was taken out with PE film, PMMA could maintain the structure of AuNPs layer.17 However, in the absence of PMMA, the assembled AuNPs layer was destroyed when it was taken out, due to the gravity and water surface tension. SEM was used to observe the structure of AuNPs layer on PE film without PMMA. It was found that the structure of AuNPs was sparse and orderless (Supporting Information Figure S5b). The corresponding UV−vis spectrum showed there was only a weak absorption band at 550 nm in the absence of PMMA (Figure 2a). This indicated that the amount of AuNPs was very low, which was consistent with the SEM observation. With the increment of PMMA concentration, the signal intensity at the peak of 550 nm gradually increased. In the meantime, a new absorption band emerged at high wavelength, which was resulted from the coupling of neighboring AuNPs in the assembled AuNPs layer. Furthermore, the intensity of this new absorption band was also positively correlated with the PMMA
The stability of the AuNPs/PMMA film and the reproducibility of SERS signal were of critical importance for its practical application as a SERS substrate for routine analysis. The stability of the AuNPs/PMMA film was investigated via measuring its SERS properties after treatment in harsh conditions. The obtained results showed SERS signals of MGITC adsorbed onto the AuNPs/PMMA film did not change obviously, although the AuNPs/PMMA film was previously immersed in acid (1 M HCl), or alkali (1 M NaOH), or heating at 110 °C for 1 h (Supporting Information Figure S4a). Thus, it was anticipated that the AuNPs/PMMA film could be used in different testing condition. In addition, the stability of the AuNPs/PMMA film over time was also tested. The Raman signals of the same AuNPs/PMMA film just prepared and stored for 8 months did not vary much, suggesting there was no significant degradation of AuNPs/PMMA film performance after 8 months (Supporting Information Figure S4b). Batch-tobatch reproducibility of the preparation process is good. We have produced many films via this process and the Raman signal intensities were very close (Supporting Information Figure S4c). To determine the homogeneity of the AuNPs/ PMMA film, SERS spectra of the AuNPs/PMMA film immersed in 10−5 M MGITC were collected from 14 randomly selected positions. The relative standard deviation (RSD) of signal intensities at peak of 1174 cm−1 was less than 15%, D
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on self-assembly of AuNPs into orderly layer on a simultaneously newborn PMMA template. The unique properties of the AuNPs/PMMA film allow for in situ ultrasensitive SERS analysis of analytes on the surface of irregular object. Proof of concept for the in situ analytical application of the SERS substrate was provided through directly SERS detection of MG on fish surface. It is also anticipated that the developed approach can be widely used to fabricate various flexible and transparent nanodevices.
concentration. These phenomena indicated that more layers of AuNPs were trapped in PMMA template when PMMA content increased. SERS spectra of the AuNPs/PMMA film immersed in 10−5 M MGITC with the increment of PMMA concentration were recorded (Figure 2b). As discussed above, few AuNPs was adsorbed on PE film, and no ordered layer was formed in the absence of PMMA (Supporting Information Figure S5a), thus, significant enhancement of Raman signals of MGITC could not be acquired. As shown in Figure 2b, MGITC could not be detected as no PMMA was added. When 0.4 mg/cm2 PMMA was added, PMMA was able to trap more AuNPs and formed ordered AuNPs layer with creation of “hot spots”. In this way, SERS intensity of MGITC was enhanced, and the Raman characteristic bands were clearly detected. The SERS intensity of MGITC was nearly doubled as the amount of PMMA was increased from 0.4 to 0.8 mg/cm2, implying that the selfassembled AuNPs became closer and acquired stronger electromagnetic field enhancement. However, the SERS intensity was not increased notably when more PMMA (>0.8 mg/cm2) was added. These results suggested that 0.8 mg/cm2 could be the critical PMMA concentration to form a closely packed AuNPs layer. Although further increase in PMMA concentration could trap more layers of AuNPs, the signal enhancement was not remarkable as the “hot spots” formed between layers were far away from analytes.24 In a word, PMMA was an indispensable template material to retain the order and SERS activity of the self-assembled AuNPs layer. Owing to the good flexibility and transparency, it was expected that the AuNPs/PMMA film could be served as a SERS substrate for direct, rapid and ultrasensitive detection of contaminants in various objects. The authors demonstrated the applicability of the AuNPs/PMMA film as SERS substrate through in situ detection of malachite green (MG) on fish surface, which is highly toxic and often illegally added into water body to improve the survival rate of fish.25 Figure 3 showed that no evident Raman signal was recorded when a laser was directly shot onto fish immersed in 10 μM MG solution. On the contrary, if a flexible and transparent AuNPs/ PMMA film was pasted to the fish contour, a significantly Raman signal at 1174 cm−1 designated to MG was detected. This was because the exposed AuNPs layer contact closely with the residual MG on the fish and dramatically amplified the Raman signal of MG. In this way, the in situ lowest detection limit of MG in aqueous medium where fish was immersed in was found to be 0.1 μM. In addition, compared with the conventional SERS detection method, which requires dripping preconcentrated AuNPs solution directly on the surface of object to be tested, the proposed flexible AuNPs/PMMA SERS substrate could avoid contaminating the object. The AuNPs/ PMMA SERS substrate can be regenerated by simple rinsing with deionized water for 0.5 h, and the Raman signals collected from the fresh and regenerated AuNPs/PMMA SERS substrates are almost the same (Supporting Information Figure S6). More extensive and in-depth studies are expected to extend the other applications of this method, for example, detection of residual pesticides on fruit surface.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86-592-6190977. Tel: +86592-6190785. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the partial support received from the Hundred Talents Program of Chinese Academy of Sciences, the Key Project of Science and Technology Program of Fujian Province (2013H0054) and the Science and Technology Innovation and Collaboration Team Project of the Chinese Academy of Sciences.
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REFERENCES
(1) Pieczonka, N. P. W.; Aroca, R. F. Chem. Soc. Rev. 2008, 37 (5), 946−954. (2) Yang, X.; Gu, C.; Qian, F.; Li, Y.; Zhang, J. Z. Anal. Chem. 2011, 83 (15), 5888−5894. (3) Stiles, P. L.; Dieringer, J. A.; Shah, N. C.; Van Duyne, R. P. Annu. Rev. Anal. Chem. 2008, 1, 601−626. (4) Chen, C.; Hutchison, J. A.; Clemente, F.; Kox, R.; Uji-I, H.; Hofkens, J.; Lagae, L.; Maes, G.; Borghs, G.; Van Dorpe, P. Angew. Chem., Int. Ed. 2009, 48 (52), 9932−9935. (5) Banholzer, M. J.; Millstone, J. E.; Mirkin, C. A. Chem. Soc. Rev. 2008, 37 (5), 885−897. (6) Fan, M. K.; Andrade, D.F. S.; Brolo, A. G. Anal. Chim. Acta 2011, 693 (1−2), 7−25. (7) Polavarapu, L.; Liz-Marzan, L. M. Phys. Chem. Chem. Phys. 2013, 15 (15), 5288−5300. (8) Lee, C. H.; Tian, L. M.; Singamaneni, S. ACS Appl. Mater. Interfaces 2010, 2 (12), 3429−3435. (9) Lu, G.; Li, H.; Zhang, H. Chem. Commun. 2011, 47 (30), 8560− 8562. (10) Lu, G.; Li, H.; Zhang, H. Small 2012, 8 (9), 1336−1340. (11) Hasell, T.; Lagonigro, L.; Peacock, A. C.; Yoda, S.; Brown, P. D.; Sazio, P. J. A.; Howdle, S. M. Adv. Funct. Mater. 2008, 18 (8), 1265− 1271. (12) He, D.; Hu, B.; Yao, Q. F.; Wang, K.; Yu, S. H. ACS Nano 2009, 3 (12), 3993−4002. (13) Zhang, C. L.; Lv, K. P.; Cong, H. P.; Yu, S. H. Small 2012, 8 (5), 648−653. (14) Reincke, F.; Hickey, S. G.; Kegel, W. K.; Vanmaekelbergh, D. Angew. Chem., Int. Ed. 2004, 43 (4), 458−462. (15) Li, Y. J.; Huang, W. I. J.; Sun, S. G. Angew. Chem., Int. Ed. 2004, 45 (16), 2537−2539. (16) Xu, W. G.; Ling, X.; Xiao, J. Q.; Dresselhaus, M. S.; Kong, J.; Xu, H. X.; Liu, Z. F.; Zhang, J. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (24), 9281−9286.
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CONCLUSIONS In summary, we have developed a simple and rapid method to fabricate a SERS substrate, AuNPs/PMMA film, which offers good flexibility, excellent optical transparency, and high SERS activity. The preparation of the AuNPS/PMMA film was based E
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(17) Jiao, L. Y.; Fan, B.; Xian, X. J.; Wu, Z. Y.; Zhang, J.; Liu, Z. F. J. Am. Chem. Soc. 2008, 130 (38), 12612−12613. (18) Duan, H. W.; Wang, D. Y.; Kurth, D. G.; Mohwald, H. Angew. Chem., Int. Ed. 2004, 43 (42), 5639−5642. (19) Konrad, M. P.; Doherty, A. P.; Bell, S. E. J. Anal. Chem. 2013, 85 (14), 6783−6789. (20) Carpay, F. M. A.; Cense, W. Nat. Phys. Sci. 1973, 241, 20−22. (21) Yang, Y.; Matsubara, S.; Nogami, M.; Shi, J. L.; Huang, W. M. Nanotechnology 2006, 17 (11), 2821−2827. (22) Zhong, L. B.; Zhou, X.; Bao, S. X.; Shi, Y. F.; Wang, Y.; Hong, S. M.; Huang, Y. C.; Wang, X.; Xie, Z. X.; Zhang, Q. Q. J. Mater. Chem. 2011, 21 (38), 14448−14455. (23) Bin, R.; Liu, G. K.; Lian, X. B.; Yang, Z. L.; Tian, Z. Q. Anal. Bioanal. Chem. 2007, 338 (1), 29−45. (24) Ko, H.; Singamaneni, S.; Tsukruk, W. Small 2008, 4 (10), 1576−1599. (25) Sudova, E.; Machova, J.; Svobodva, Z.; Vesely, T. Med. Vet. 2007, 52 (12), 527−539.
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Self-Assembly of Au Nanoparticles on PMMA Template as Flexible, Transparent, and Highly Active SERS Substrates Lu-Bin Zhong,† Jun Yin,† Yu-Ming Zheng,*,† Qing Liu,† Xiao-Xia Cheng,† and Fang-Hong Luo‡ †
Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Road, Xiamen 361021, P. R. China Cancer Research Center, Medical College, Xiamen University, 422 South Siming Road, Xiamen 361005, P. R. China
‡
S Supporting Information *
ABSTRACT: We report a simple and rapid method for fabricating a surface-enhanced Raman scattering (SERS) substrate, which offers good flexibility, excellent optical transparency, and high SERS activity. Specifically, the SERS substrate (AuNPs/PMMA film) was obtained through self-assembly of gold nanoparticles (AuNPs) on newborn poly(methyl methacrylate) (PMMA) template. The UV−vis spectroscopy analysis and scanning electron microscopy observation revealed that the gold nanoparticles were closely assembled on the flexible and transparent PMMA template. The fabricated AuNPs/PMMA film SERS substrate allowed detection of model molecule, malachite green isothiocyanate, at a concentration as low as 0.1 nM, and exhibited good reproducibility in the SERS measurement. The Raman enhancement factor (EF) of the AuNPs/PMMA film was found to be as high as (2.4 ± 0.3) × 107. In addition, measure of residual malachite green on fish surface was carried out, and the result indicated that the AuNPs/PMMA film had great potential in the in situ ultrasensitive detection of analyte on irregular objects.
S
detached from the paper because of the weak physical adsorption. To firmly fix the nanoparticles on the templates, usually chemical modifications of nanoparticles or templates were required, which was rather tedious.9,10 To save the process of chemical modification, Hasell et al. took advantage of the physical constraint of the polymer template to fix nanoparticles.11 Unfortunately, this method requires 24 h heating under high pressure to dissolve the precursor complex and infuse it into the polymer. Recently, Yu et al. presented a new class of SERS substrates fabricated via electrospinning technique.12,13 However, because the nanoparticles were incorporated inside opaque nanofibers, which resulted in the reduction of in situ detection sensitivity. Hence, it is an urgent need to develop a simple, rapid and cost-efficient way for fabrication of flexible SERS substrate. Herein, we present a novel fabrication strategy to produce highly active SERS substrates with good flexibility and optical transparency. More importantly, the SERS substrates can be easily and quickly prepared without any expensive instrument. The authors demonstrate the method for constructing flexible SERS substrates through self-assembly of Au nanoparticles (AuNPs) in newborn poly(methyl methacrylate) (PMMA) template, which is denoted as AuNPs/PMMA film. The preparation procedure is shown in Scheme 1.
urface-enhanced Raman scattering (SERS) has been recognized as a powerful molecular spectroscopic technique, which allows nondestructive chemical or biochemical analysis with ultrasensitive detection.1,2 Two mechanisms have been widely accepted to account for the SERS effect, chemical enhancement, and electromagnetic enhancement. Compared to chemical enhancement, the strong electromagnetic field enhancement near metallic nanoparticles is mainly responsible for the occurrence of SERS.3,4 For this reason, research interests are turning toward developing advanced metal nanoparticle-based SERS substrates, which are capable of generating high-quality Raman scattering signal from interested analytes.5,6 However, most of works were devoted to fabricate SERS substrates with hard templates (such as glass, quartz, or silicon). Because of lack of flexibility, the target analytes on irregular shaped objects need to be extracted with suitable solvent before SERS analyzing, which limits its application to in situ detection of analytes on planar objects only.7 Therefore, there is a strong need and has an increasing interest in the development of flexible, transparent and highly active SERS substrates, which are capable of providing in situ ultrasensitive SERS analysis of analytes on objects with diverse morphologies.7 Although there are some reports on the preparation of flexible SERS substrates, the methods have some drawbacks. For example, Lee et al. reported the fabrication of flexible SERS substrates by dipping a paper into the nanoparticles solution.8 The method was simple, but it required a rather concentrated nanoparticle solution, and the nanoparticles were easily © XXXX American Chemical Society
Received: December 28, 2013 Accepted: May 29, 2014
A
dx.doi.org/10.1021/ac404224f | Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Article
Poly(methyl methacrylate) (PMMA, MW = 1.2 × 105) was purchased from Sigma-Aldrich. Malachite green isothiocyanate (MGITC) was purchased from Invitrogen Corporation, Carlsbad, CA. Fish were purchased from local supermarket. Synthesis of AuNPs. AuNPs were prepared according to the method reported by Carpay and Cense with minor modification.20 In a typical synthesis, 1 mL of sodium citrate solution (38.8 mM) was quickly added to 99 mL of aqueous HAuCl4 solution (0.243 mM) that was stirred in boiling condition, and the mixture was boiled for 5 min to give a winered solution. Another 1 mL of sodium citrate solution (38.8 mM) and 1 mL of HAuCl4 solution (0.243 mM) were then successively added to the mixture every 20 s, and this step was repeated by 3 times. After that, heating was stopped, and the reaction mixture was allowed to cool down to room temperature. The mean size of the prepared AuNPs was 55 ± 5 nm. Fabrication of AuNPs/PMMA SERS Substrate. First, 15 mL of freshly prepared AuNPs suspension was transferred to a beaker, followed by addition of 7 mL of toluene with different concentrations of PMMA (0−3 mg/cm2). Because of the immiscibility of water and toluene, a clear water/toluene interface was formed. After that, 8 mL of ethanol was injected into the AuNPs suspension using a mechanical syringe pump (LSPO4-1A, Baoding Longer Precision Pump Co., Ltd.) with a feeding rate of 10 mL/h. With the addition of ethanol, the AuNPs rose up to the water/toluene interface and selfassembled into orderly AuNPs layers. A thin PMMA template was simultaneously formed on the top of the aqueous solution because of the evaporation of toluene. The self-assembled AuNPs layer was then fixed on the newborn PMMA template. When the toluene fully evaporated, a polyethylene (PE) film was used as a carrier to retrieve the prepared AuNPs/PMMA film from the top of the aqueous solution surface. Finally, the PE film carried AuNPs/PMMA film was dried at 60 °C in a vacuum oven, and used as SERS substrate in the subsequent experiments. The PE film was found to be fastened tightly to the AuNPs/PMMA film, and was not detached from the AuNPs/PMMA film in the SERS detection process. The preparation process did not involve tedious procedures and expensive equipment, and the whole process can be completed within a few hours. Preparation of SERS Samples. To determine SERS enhancement factor, SERS sample 1 was prepared as follows: The prepared AuNPs/PMMA film was immersed in 10−5 M MGITC ethanol solution for 20 min. The AuNPs/PMMA film was then washed with ethanol to remove the unbound MGITC molecules and dried at room temperature to evaporate all of the ethanol. To evaluate the applicability of the prepared AuNPs/PMMA film as a SERS substrate for in situ analyte detection, SERS sample 2 was prepared. Fish were immersed in solutions with different MGITC concentrations for 10 min. The AuNPs/ PMMA film was wetted with water and the side exposing AuNPs was attached to fish surface for direct SERS measurement. Characterization. Optical characterization was carried out by a UV−vis spectrophotometer (UV-759, China). SEM images were obtained with a field-emission scanning electron microscope (Hitachi S-4800, Japan). SERS spectra were recorded using a confocal microscope Raman spectrometer (LabRAM Aramis, France). A 785 nm semiconductor diode laser was used for Raman excitation, which was focused onto a
Scheme 1. Schematic Representation of Preparation of AuNPs/PMMA SERS Substratea
a
Typical photographs of the prepared SERS substrate are demonstrated in the top right corner.
First, AuNPs were well dispersed in aqueous solution, and then PMMA toluene solution was added gently to the top of the AuNPs solution. After that, ethanol was added dropwise to the AuNPs solution and induced the AuNPs to rise slowly to the water/toluene interface. With the addition of ethanol, more and more AuNPs rose up and self-assembled into orderly layer with innumerable “hot spots”.14,15 In the meantime, the PMMA molecules built up at the water/toluene interface to form a thin PMMA template because of the evaporation of toluene. The newborn thin PMMA film served as a template to fix the selfassembled AuNPs layer by the physical constraint. One side of the AuNPs layer was tightly wrapped by the PMMA template, the opposite side was unattached and exposed to the aqueous solution. It is expected that the exposed AuNPs layer can directly contact with analytes and enhance the characteristic Raman signal maximally. After the complete evaporation of toluene, a polyethylene (PE) film was used as a carrier to retrieve the thin AuNPs/ PMMA film from the top of aqueous solution, as the AuNPs/ PMMA film is too thin to be taken out. The whole process can be completed in a short time without any expensive equipment. PMMA was chosen as an ideal template polymer for fixing the self-assembled AuNPs layers because of its excellent optical transparency. Light can pass through PMMA template and reach AuNPs layer to activate the plasmon resonance, which gives rise to the enormous SERS enhancement. Moreover, because of PMMA has a low Raman cross-section, the AuNPs/ PMMA film is expected to exhibit clear SERS signals.16,17 As shown in the photographs of Scheme 1, the AuNPs/PMMA film can be bended sharply, implying its good flexibility. The side of AuNPs layer of AuNPs/PMMA film exhibits metallic luster because of electronic coupling of closely packed AuNPs.18,19 The other side of AuNPs/PMMA film (PMMA template layer) is transparent, and laser can go through it to reach the AuNPs layer, herein we define the AuNPs/PMMA film as a transparent SERS substrate.
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EXPERIMENTAL SECTION Chemical Reagents. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4 ·3H 2 O, 99.9%), sodium citrate (C 6 H 5 Na 3 O 7 , 99%), ethanol (C 2 H 6 O, 99%), toluene (C6H5CH3, 99%), and malachite green (C23H25CIN2) were purchased from Sinopharm Chemical Reagent Co., Ltd. B
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Analytical Chemistry
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Figure 1. (a) UV−vis spectra of monodispersed AuNPs, AuNPs/PMMA film and MGITC solution. (The inset is a SEM image of the AuNPs/ PMMA film.) (b) SERS spectra of the AuNPs/PMMA film immersed in different concentration of MGITC solutions. (c) SERS spectra of MGITC collected from both sides of the AuNPs/PMMA film. Experimental conditions: the AuNPs/PMMA film containing 0.8 mg/cm2 PMMA; Raman reporter molecule is MGITC; laser wavelength =7 85 nm; laser power on the sample = 0.2 mW; integration time = 5 s.
sample with a spot size of approximately 3 μm2. The SERS measurements were performed in a confocal microscope using a 50× objective (Leica) with a numerical aperture of 0.55.
enhancement is mainly attributed to surface-enhanced Raman scattering instead of resonance enhancement.22 The Raman enhancement factor (EF) was used to evaluate the SERS activity of the prepared AuNPs/PMMA film quantitatively. The formula of EF is shown as below.23
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RESULTS AND DISCUSSION The UV−vis spectra in Figure 1a showed that the monodispersed AuNPs displayed a strong characteristic band at 550 nm, while the AuNPs/PMMA film exhibited two absorption bands. A peak at 558 nm was near to the absorption band of monodispersed AuNPs, and the other one was around 900 nm. The new absorption band can be ascribed to strong coupling between adjacent AuNPs, indicating the formation of assembled AuNPs layers on the PMMA template.21 SEM observation confirmed this hypothesis. The inset in Figure 1a showed that AuNPs were self-assembled into highly ordered layers in PMMA, and numerous “hot spots” had formed, which could significantly amplify Raman signal of analytes adsorbed on its surface (More SEM images with different magnification were provide in Supporting Information Figure S1). Although PMMA was close to AuNPs, due to its weak Raman signal, it gave a rather “clean” Raman background even after enhancement by the assembled AuNPs (see details in section 2 in the Supporting Information). To assess the detection sensitivity of the AuNPs/PMMA film, SERS spectra of AuNPs/PMMA films immersed in different concentrations of MGITC were measured. As shown in Figure 1b, the Raman characteristic bands of MGITC were clearly shown even when the MGITC concentration was reduced to 0.1 nM. Figure 1a showed the characteristic peak of MGITC UV−vis spectrum was 620 nm, which was far away from the excitation wavelength at 785 nm. Thus, Raman
EF =
ISERS/NSERS cN σhI ≈ A SERS INRS/NNRS RINRS
(1)
The obtained result showed that the EF of AuNPs/PMMA film was as high as (2.4 ± 0.3) × 107 (see details in section 3 in the Supporting Information). Besides, two other molecules, 4aminothiophenol and 4-mercaptopyridine were also used to evaluate Raman EF of the AuNPs/PMMA film under the same condition. The results showed that they had similar EF, (3.6 ± 0.5) × 107 and (9.0 ± 1.0) × 107, respectively, indicating that the AuNPs/PMMA film was able to greatly enhance the Raman signal of other components (Supporting Information Figure S3). As PMMA possesses excellent optical transparency, we presume that laser can easily pass through PMMA template and reach the assembled AuNPs layer. On this occasion, plasmon resonance of the assembled AuNPs layer would be motivated, and then the local electromagnetic field could be greatly enhanced, which markedly amplifies the Raman signal of analytes adsorbed on AuNPs layer surface.4 As shown in Figure 1c, the SERS intensities collected from both sides of the AuNPs/PMMA film were almost identical, which verified our supposition. Hence, it was expected that the SERS signal directly collected from the side of PMMA would not be sacrificed when the film was wrapped over an object surface. C
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Figure 2. (a) UV−vis spectra of the AuNPs/PMMA SERS substrate with the increment of PMMA concentration. (b) SERS spectra of the AuNPs/ PMMA SERS substrate immersed in 10−5 M MGITC with the increment of PMMA concentration. Laser wavelength = 785 nm; laser power on the sample = 0.2 mW; integration time = 5 s.
Figure 3. In situ detection of MG on fish surface. Experimental conditions: Laser wavelength = 785 nm; laser power on the sample = 2 mW; integration time = 20 s.
indicating a good homogeneity of the film (Supporting Information Figure S4d). A noteworthy fact was that PMMA content significantly affected the structure and property of the assembled AuNPs layer. If there was no PMMA in toluene, the assembled AuNPs at the water/toluene interface was unable to form complete AuNPs layers on the PE film (Supporting Information Figure S5a). The reason may be that PMMA serve as a template to fix the assembled AuNPs layers. When the assembled AuNPs layer was taken out with PE film, PMMA could maintain the structure of AuNPs layer.17 However, in the absence of PMMA, the assembled AuNPs layer was destroyed when it was taken out, due to the gravity and water surface tension. SEM was used to observe the structure of AuNPs layer on PE film without PMMA. It was found that the structure of AuNPs was sparse and orderless (Supporting Information Figure S5b). The corresponding UV−vis spectrum showed there was only a weak absorption band at 550 nm in the absence of PMMA (Figure 2a). This indicated that the amount of AuNPs was very low, which was consistent with the SEM observation. With the increment of PMMA concentration, the signal intensity at the peak of 550 nm gradually increased. In the meantime, a new absorption band emerged at high wavelength, which was resulted from the coupling of neighboring AuNPs in the assembled AuNPs layer. Furthermore, the intensity of this new absorption band was also positively correlated with the PMMA
The stability of the AuNPs/PMMA film and the reproducibility of SERS signal were of critical importance for its practical application as a SERS substrate for routine analysis. The stability of the AuNPs/PMMA film was investigated via measuring its SERS properties after treatment in harsh conditions. The obtained results showed SERS signals of MGITC adsorbed onto the AuNPs/PMMA film did not change obviously, although the AuNPs/PMMA film was previously immersed in acid (1 M HCl), or alkali (1 M NaOH), or heating at 110 °C for 1 h (Supporting Information Figure S4a). Thus, it was anticipated that the AuNPs/PMMA film could be used in different testing condition. In addition, the stability of the AuNPs/PMMA film over time was also tested. The Raman signals of the same AuNPs/PMMA film just prepared and stored for 8 months did not vary much, suggesting there was no significant degradation of AuNPs/PMMA film performance after 8 months (Supporting Information Figure S4b). Batch-tobatch reproducibility of the preparation process is good. We have produced many films via this process and the Raman signal intensities were very close (Supporting Information Figure S4c). To determine the homogeneity of the AuNPs/ PMMA film, SERS spectra of the AuNPs/PMMA film immersed in 10−5 M MGITC were collected from 14 randomly selected positions. The relative standard deviation (RSD) of signal intensities at peak of 1174 cm−1 was less than 15%, D
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on self-assembly of AuNPs into orderly layer on a simultaneously newborn PMMA template. The unique properties of the AuNPs/PMMA film allow for in situ ultrasensitive SERS analysis of analytes on the surface of irregular object. Proof of concept for the in situ analytical application of the SERS substrate was provided through directly SERS detection of MG on fish surface. It is also anticipated that the developed approach can be widely used to fabricate various flexible and transparent nanodevices.
concentration. These phenomena indicated that more layers of AuNPs were trapped in PMMA template when PMMA content increased. SERS spectra of the AuNPs/PMMA film immersed in 10−5 M MGITC with the increment of PMMA concentration were recorded (Figure 2b). As discussed above, few AuNPs was adsorbed on PE film, and no ordered layer was formed in the absence of PMMA (Supporting Information Figure S5a), thus, significant enhancement of Raman signals of MGITC could not be acquired. As shown in Figure 2b, MGITC could not be detected as no PMMA was added. When 0.4 mg/cm2 PMMA was added, PMMA was able to trap more AuNPs and formed ordered AuNPs layer with creation of “hot spots”. In this way, SERS intensity of MGITC was enhanced, and the Raman characteristic bands were clearly detected. The SERS intensity of MGITC was nearly doubled as the amount of PMMA was increased from 0.4 to 0.8 mg/cm2, implying that the selfassembled AuNPs became closer and acquired stronger electromagnetic field enhancement. However, the SERS intensity was not increased notably when more PMMA (>0.8 mg/cm2) was added. These results suggested that 0.8 mg/cm2 could be the critical PMMA concentration to form a closely packed AuNPs layer. Although further increase in PMMA concentration could trap more layers of AuNPs, the signal enhancement was not remarkable as the “hot spots” formed between layers were far away from analytes.24 In a word, PMMA was an indispensable template material to retain the order and SERS activity of the self-assembled AuNPs layer. Owing to the good flexibility and transparency, it was expected that the AuNPs/PMMA film could be served as a SERS substrate for direct, rapid and ultrasensitive detection of contaminants in various objects. The authors demonstrated the applicability of the AuNPs/PMMA film as SERS substrate through in situ detection of malachite green (MG) on fish surface, which is highly toxic and often illegally added into water body to improve the survival rate of fish.25 Figure 3 showed that no evident Raman signal was recorded when a laser was directly shot onto fish immersed in 10 μM MG solution. On the contrary, if a flexible and transparent AuNPs/ PMMA film was pasted to the fish contour, a significantly Raman signal at 1174 cm−1 designated to MG was detected. This was because the exposed AuNPs layer contact closely with the residual MG on the fish and dramatically amplified the Raman signal of MG. In this way, the in situ lowest detection limit of MG in aqueous medium where fish was immersed in was found to be 0.1 μM. In addition, compared with the conventional SERS detection method, which requires dripping preconcentrated AuNPs solution directly on the surface of object to be tested, the proposed flexible AuNPs/PMMA SERS substrate could avoid contaminating the object. The AuNPs/ PMMA SERS substrate can be regenerated by simple rinsing with deionized water for 0.5 h, and the Raman signals collected from the fresh and regenerated AuNPs/PMMA SERS substrates are almost the same (Supporting Information Figure S6). More extensive and in-depth studies are expected to extend the other applications of this method, for example, detection of residual pesticides on fruit surface.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86-592-6190977. Tel: +86592-6190785. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the partial support received from the Hundred Talents Program of Chinese Academy of Sciences, the Key Project of Science and Technology Program of Fujian Province (2013H0054) and the Science and Technology Innovation and Collaboration Team Project of the Chinese Academy of Sciences.
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REFERENCES
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CONCLUSIONS In summary, we have developed a simple and rapid method to fabricate a SERS substrate, AuNPs/PMMA film, which offers good flexibility, excellent optical transparency, and high SERS activity. The preparation of the AuNPS/PMMA film was based E
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