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Preparation of gold nanoparticles–agarose gel composite and its application in SERS detection
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 657–661
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Preparation of gold nanoparticles–agarose gel composite and its application in SERS detection Xiaoyuan Ma a,b,c, Yu Xia a,c, Lili Ni a, Liangjing Song a, Zhouping Wang a,c,⇑ a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China The State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, PR China c Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, PR China b
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
Nanocomposites of agarose gel
embedded with gold nanoparticles were prepared. Dynamic hot-spots were generated from the agarose gel contraction loss of water. The nanocomposites were used successfully to detect Raman signal molecules. Recycling of the nanocomposites could be achieved with washing solution.
a r t i c l e
i n f o
Article history: Received 4 July 2013 Received in revised form 25 November 2013 Accepted 28 November 2013 Available online 7 December 2013 Keywords: Agarose gel Gold nanoparticles Nanocomposites SERS
a b s t r a c t Agarose gel/gold nanoparticles hybrid was prepared by adding gold nanoparticles to preformed agarose gel. Nanocomposite structures and properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and UV–Vis–NIR absorption spectroscopy. Based on the swelling–contraction characteristics of agarose gel and the adjustable localized surface plasmon resonance (LSPR) of the gold nanoparticles, the nanocomposites were used as surface enhanced Raman scattering (SERS) substrate to detect the Raman signal molecules (NBA, MBA, 1NAT). Results revealed that the porous structure of the agarose gel provided a good carrier for the enrichment of the gold nanoparticles. The gold nanoparticles dynamic hot-spot effect arising from the agarose gel contraction loss of water in the air greatly enhanced the Raman signal. Furthermore, the gel could be cleaned with washing solution and recycling could be achieved for Raman detection. Ó 2013 Elsevier B.V. All rights reserved.
Introduction In recent years, nanotechnology has received great advances in different research areas. Nanomaterials have been widely used in catalysis, biosensing detection and other fields due to its small size effect, high specific surface area and enhanced optical properties (quantum dot fluorescence effect, the quenching effect of gold material and surface-enhanced Raman properties, etc.) [1–3]. ⇑ Corresponding author. Tel./fax: +86 510 85917023. E-mail address: [email protected] (Z. Wang). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.11.111
Recently, a new functional material combined with nanostructures introduced into polymers has received widespread research interests. This kind of composite materials possess the advantages of both the nanostructures and the polymers which exhibit excellent performances. For example, with the combination of gold nanoparticles (GNPs) and TiO2, the photocatalysis properties could be greatly enhanced [4]. Up to now, nanocomposites have made great progress in sensors, microelectronic components, biochemical engineering (e.g. gene sequencing), chemical catalysts and other applications [5–8].
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X. Ma et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 657–661
Agarose is a kind of straight-chain polysaccharide extracted from seaweed or other vegetables. On heating to above 90 °C, the agarose powder is generally dissolved in water. When the temperature dropped to 35–40 °C, a good semi-solid gel will be formed. This is the main feature and foundation for a variety of usage. The gel gelation characteristics created by the presence of hydrogen bonds can be destroyed by any factor lead to the destruction of hydrogen bonds. The pore size of agarose could be changed by the concentration of agarose powder. It is because of this particular gelling properties combined with the significant stability, shrink and swell features as well as the easy absorption behavior, agarose gel has been widely used in food, pharmaceutical, chemical, textile, and other fields [9–11]. The surface-enhanced Raman scattering (SERS) phenomenon was discovered for more than 30 years of history. However, the spectrum is so weak to detect and the applications are limited. With the development of materials chemistry and instrument technology, especially the increased demand for trace detection, SERS has become one of the hot researches now. Substrate materials with a specific structure can effectively enhance the SERS signal. Here, nanomaterials with the high adjustable structural characteristics (size, shape, spacing, etc.) are excellent SERS substrates. Among them, the gold, silver nanomaterials due to their good optical absorption features are widely used in SERS studies. Owing to the high sensitivity and specificity of SERS technology, it has been widely applied in a majority of areas, including security, food safety monitoring, clinical diagnostics, trace analysis and biochemical analysis [12–15]. In this paper, the agarose gel was used as a template for the adsorption of prepared colloidal GNPs. The morphology and optical properties of the composite film were characterized. Combined with the shrink–swell properties of agarose gel and the optical absorption properties of GNPs, the composite film could be used as a good SERS substrate for the detection of Raman signal molecules, such as Niel blue A sulfate (NBA), 4-mercaptobenzoic acid (MBA), 1-naphthalenethiol (1NAT). The SERS spectra gradually increased due to the ‘‘hot spot effect’’ generated upon dehydration process when the agarose gel was exposed to the air as the network volume decreased and the GNPs got close to each other. Additionally, Raman molecules could be washed out when immersed in a cleaning solution, so that recycling could be achieved. Experimental details Materials Hydrogen tetrachloroaurate trihydrate (HAuCl44H2O), sodium borohydride (NaBH4), potassium carbonate (K2CO3), sodium citrate (C6H5Na3O7) were purchased from Shanghai Chemical Reagent Co., Ltd., China. Agarose was purchased from Sangon Biotech (Shanghai) Co., Ltd., NBA, MBA, 1NAT were purchased from Sigma–Aldrich. All the chemical reagents were of analytical grade or higher and used without further purification. Aqueous solutions used in the experiments were prepared using Milli-Q water from Milli-Q system (resistivity > 18 MX). All procedures were conducted at room temperature unless otherwise specified. Synthesis of GNPs GNPs (5 nm in diameter) were prepared by the reduction of AuCl4 with NaBH4 according to literature procedures [16]. Typically, an aqueous solution of HAuCl4 (200 mL, 36.4 mM) was first mixed with an aqueous solution of K2CO3 (1 mL, 0.2 M), and then reduced by the quick addition of a freshly prepared solution of NaBH4 (9 mL, 0.5 mg/mL1). The mixture turned wine red rapidly,
indicating the generation of GNPs. The obtained solution of GNPs was stored at 4 °C until use. Preparation of agarose gels Briefly, agarose hydrogels (1.5% w) were prepared by dissolving 225 mg of agarose powder in 15 mL of water and heated in microwave until complete dissolution to transparency and immediately poured into a ‘‘U’’ mold of 3 mm thickness and stored for at least 1 h at 4 °C. Then, the slab gel was cut into small films having a diameter of 1 cm and thickness of 3 mm using the hole puncher. Agarose gel film was immersed in ultrapure water and oscillated to remove the dispersed gel pieces. Synthesis of the gold nanoparticles–loaded agarose gel (GNPs– Agarose) Agarose gel film was immersed in a solution of GNPs. The GNPs could be spread to the gel membrane reticular formation to form GNPs–Agarose composite film. The corresponding UV–Vis–NIR absorption peak of the film was measured every once in a while and the peak position and intensity was recorded. SERS measurement NBA, MBA and 1NAT were selected as the Raman signal molecules (5 lM). Different concentrations of NBA were used to do the sensitivity detection ranging from 5 108 to 5 1010 M. The GNPs–Agarose composite film was immersed in a certain concentration of the solution in the NBA, MBA, 1NAT for the sufficient adsorption of probe molecules. Then the Raman absorption peak was measured in Raman spectroscopy with a Renishaw Invia Reflex system equipped with Peltier-cooled charge-coupled device (CCD) detectors and a Leica confocal microscope. Samples were excited with a 785 nm diode laser under linefocus mode and a grating of 1200 mm1 was used. The corresponding laser was focused onto the sample surface using a 50 long working distance objective. Spectra were collected in continuous mode with 10 s exposure time and accumulated for 2 times, the laser power was adjust to 0.05% which was about 0.06 mW. The changes of absorption peak were recorded over time. For reversible experiments, the GNPs– Agarose composite film was first immersed in analyte and the SERS spectra were recorded. Then, the film was washed by 1% sodium citrate solution and the SERS spectrum was again recorded. This procedure was repeated for at least 3 times. Results and discussion Preparation and characterization of GNPs–Agarose GNPs were prepared by the reduction of HAuCl4 using NaBH4 which called colloidal reduction chemical method. K2CO3 was added in the reaction system as a stabilizer and the protecting agent. Therefore, GNPs could be uniformly dispersed and stably present in the aqueous solution. Fig. 1(a and b) shows the TEM images and UV–Vis–NIR absorbance spectra of GNPs. GNPs were of spherical morphology with the diameter of 5 nm. And the plasmon peak was at 508 nm. The plasmon peak did not change apparently with time changes which illustrated that the gold colloid could be existed stably in solution. After the GNPs were attached to the agarose gel network, the morphological images and absorbance spectra were shown in Fig. 1(c and d). It can be seen that the GNPs were evenly distributed in the agarose gel film and were separated by a distance of gaps. The agarose gel network size and the mechanical strength can be adjusted by changing the
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Fig. 1. TEM images (a) and UV–Vis–NIR absorbance spectra (b) of GNPs. The morphological images (c) and absorbance spectra upon time from 1 h, 4 h, 8 h, 12 h, 15 h, 18 h, 20 h, 24 h respectively (d) of GNPs–Agarose gel.
concentration of the agarose gel. The agarose powder was dissolved in boiling water and then formed the porous structure under the cooling process. It could be used effectively as a natural porous template for the enrichment of the colloidal GNPs or other nanomaterials as a synthetic method for the composite structures combined with nanomaterials and organic materials. The absorbance spectra for GNPs–Agarose was slightly red shifted to 518 nm compared with the GNPs in solution. The LSPR absorbance spectra of the metal nanomaterials was affected by its morphology, particle size, degree of aggregation, environmental media refractive index and many other factors [17]. The red shift could be ascribed to the changes of the environmental media refractive index that the GNPs were first dispersed simply in water and then among the agarose gel network [18]. With the immersion time went on, more GNPs adsorbed to the gel membrane which made the absorbance intensity gradually increased. At the same time, some GNPs might stack to each other and resulted in the absorption peaks slightly red-shifted toward the long wavelength. By comparing the absorbance spectra of GNPs and the GNPs–Agarose composite gel in Fig. 1, we can see that about 90% GNPs were embedded in the network of agarose gel. So, about 1.97 1016 GNPs were loaded in the agarose gel film under the experimental conditions (suppose the diameter of GNPs was about 4 nm in average). SERS effect of GNPs–Agarose The agarose gel network loaded with high density of GNPs combined the swelling characteristic of the gel and the strong optical absorbance (localized surface plasmon resonance) of GNPs which made the composite materials good substrate in SERS detection. The enhanced SERS effect provided by these materials is related to the generation of a high density of hot spots when the gel
collapses upon dehydration. The consequent volume reduction of the materials drives the embedded gold colloidal nanoparticles close to each other, thus prompting the interaction between their respective electromagnetic fields and therefore further increasing the enhanced Raman signal. Fig. 2(a) shows the background SERS spectra of the dry, unloaded (with no Raman signal analyte) GNPs–Agarose and GNPs– Agarose with NBA molecules. Results presented a clean spectral window for the blank GNPs–Agarose gel. In contrast, upon immersion of the gel in an aqueous solution of NBA and subsequent airdrying, the SERS spectra showed well-defined bands with high intensity which are characteristics of NBA, including 592 cm1, 1141 cm1, 1351 cm1, 1429 cm1, 1492 cm1, 1544 cm1 and 1640 cm1. The uniformity of the entire surface was shown in Fig. 2(b) with eight Raman spectra at different detection point. It can be seen clearly that the Raman spectra remained almost the same at different detection area. The characteristic Raman shifts of NBA at 592 cm1 were used to calculate the relative standard deviation (RSD). A value of 5.44% for RSD was received which showed that the SERS enhancing ability of the GNPs–Agarose gel was homogeneous through the entire surface. It is an efficient and sensitive substrate for SERS detection. The dynamic hot spots induced SERS effect was demonstrated by using NBA as signal molecule during the dehydration process of the GNPs–Agarose gel. Fig. 3(a) depicts the substantial increase of the Raman signal. When the composite film loaded with Raman active molecule exposed to the air, the composite film dehydrated and shrinkaged. The reduced volume caused the GNPs closed to each other which resulted in a ‘‘dynamic hot spot effect’’ [19]. With time went by from 0 min to 120 min, the Raman peak appeared a 20-fold increase. This Raman signal enhancement effect may be caused by the following reasons. Firstly, in our experiment, when the agarose gel shrinked upon dehydration process, the imbedded
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Fig. 2. (a) SERS spectra of the dry GNPs–Agarose gel with (black line) and without (red line) NBA Raman molecule. (b) SERS spectra for uniformity of the entire GNPs–Agarose gel. (Eight random points were measured). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. (a) SERS spectra of GNPs–Agarose gel adsorbed with NBA Raman molecule upon dehydration on air from 0 min to 120 min. (b) SERS spectra obtained with different concentrations of NBA (a) 5 108 M, (b) 5 109 M, (c) 5 1010 M; (d) the spectrum measured while a non-SERS substrate was immersed in a solution with NBA concentration of 5 102 M.
GNPs got closer to each other which enhanced the aggregation extent and thereby generating the high electromagnetic fields. The enhanced LSPR features may cause the SERS intensity increasement [20]. And the distance between NBA molecules also decreased along with the dehydration process. So the number of NBA molecules at each unit laser spot increased which might also lead to SERS enhancement. Then, since the volume shrinkage of the composite gel causes the distance between GNPs decreases, the coupling between each other will have a ‘‘hot spot effect’’ [21– 23]. Because the surface enhancement factor (EF) is found to have many definitions in published literatures [24,25], in this manuscript, it is calculated by using the analytical chemistry point of view through the analytical EF (AEF) defined as [25]:
AEF ¼ ðISERS =C SERS Þ=ðIRS =C RS Þ where ISERS corresponds to the Raman intensity obtained for the SERS substrate under a certain concentration CSERS and IRS corresponds to the Raman intensity obtained under non-SERS conditions at an analyte concentration of CRS. According to this established definition, the AEF of the substrate was estimated by considering the 592 cm1 Raman shift, because it is the strongest peak of all bands in the spectra. When using CSERS = 5 109 M and CRS = 5 102 M and the intensities obtained from Fig. 3(b), we can obtain a value of about AEF = 107. It should be noted that the AEF is different when different Raman band or CSERS are chosen for estimation. But those AEF values are the same order of magnitude. Besides, we have compared the SERS substrate for GNPs–Agarose composite film and dispersed GNPs. Results (shown in Fig. S1) revealed that when the
agarose gel was wet before dehydration, the Raman intensity for GNPs–Agarose was slightly higher than GNPs themselves. While the agarose gel was collapsed upon dehydration, the Raman intensity was significantly enhanced. These data illustrated that the GNPs–Agarose film is superior than dispersed GNPs as SERS substrate. Fig. 3(b) shows the obtained SERS spectra when NBA solutions were added with different concentrations ranging from 5 108 to 5 1010 M. 5 1011 M NBA did not show any characteristic peaks, and thus the 5 1010 M concentration is taken as the limit of detection for the NBA molecule. The reversibility of the GNPs–Agarose gel was also studied for NBA and the results were shown in Fig. 4. The gel was first immersed in the solution containing the analyte, characterized by SERS, and then immersed in a washing aqueous solution of 1% sodium citrate, and the SERS spectra were measured again. This process was repeated three times to ensure the full reusability of this sensor platform. For NBA molecules, it is very likely that sodium and citrate ions compete for the retention sites of the analyte on the nanoparticles, thus displacing it because of their much higher concentration and thereby cleaning the sensor. When the analyte was again in contact with the gel, it was retained, giving rise to signals of similar intensity. As for other Raman signal molecules, for example, an anion (MBA) and a neutral molecular species (1NAT), the Raman spectra were obtained as shown in Fig. S2. When the GNPs–Agarose gel dehydrated in air, the Raman signal increased significantly. The same spectra changes could be explained as for Fig. 3. The
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of China (2012BAK08B01), the S&T Supporting Project of Jiangsu (BE2011621, BE2012614), funding from the State Key Laboratory of Bioelectronics of Southeast University. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.11.111. References
Fig. 4. Reversible SERS spectra of GNPs–Agarose gel after adsorbed with NBA Raman molecule (dry film) and washed with sodium citrate solution for three times. (The upper line is the gel with analyte and the lower line is the gel washed with sodium citrate).
reversibility experimental results (Fig. S3) revealed that the composite films could also be reused for anion and neutral molecules detection. Conclusion The agarose gel-based porous structures were used as template to adsorb prepared GNPs. The morphological and optical properties of the composite GNPs–Agarose gel hybrid were characterized and analyzed using TEM, SEM and UV–Vis–NIR. When the composite film was exposed to the air, the agarose gel dehydrated and shrinked. The GNPs got close to each other and the hot-spot effect generated. Results revealed that this composite film could be used as an effective SERS substrate for the detection of Raman signal molecules (NBA, MBA, 1NAT). The SERS signal gradually increased upon the dehydration process due to the dynamic hot-spot effect. Additionally, this composite material could be cleaned by washing solution and be reused when immersed in Raman molecules. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grant 21375049), National S&T Support Program
[1] Y.Y. Broza, H. Haick, Nanomedicine 8 (2013) 785–806. [2] A. Elbakry, E.C. Wurster, A. Zaky, R. Liebl, E. Schindler, P. Bauer-Kreisel, T. Blunk, R. Rachel, A. Goepferich, M. Breunig, Small 8 (2012) 3847–3856. [3] B.M. Popescua, N. Alib, G. Bastureac, G.I. Comsaa, L.A. Materond, M. Chipara, Appl. Surf. Sci. 275 (2013) 2–6. [4] S. Ajaikumar, M. Golets, W. Larsson, A. Shchukarev, K. Kordas, A.R. Leino, J.P. Mikkola, Microporous Mesoporous Mater. 173 (2013) 99–111. [5] M. Ahmed, R. Narain, Prog. Polym. Sci. 38 (2013) 767–790. [6] R.S. Dey, R.K. Bera, C.R. Raj, Anal. Bioanal. Chem. 405 (2013) 3431–3448. [7] G.Q. Jian, N.W. Piekiel, M.R. Zachariah, J. Phys. Chem. C 116 (2012) 26881– 26887. [8] A.C. Ferrandez, S. Baranton, J. Bigarre, P. Buvat, C. Coutanceau, Langmuir 51 (2012) 17832–17840. [9] N. Fatin-Rouge, A. Milon, J. Buffle, R.R. Goulet, A. Tessier, J. Phys. Chem. B 107 (2003) 12126–12137. [10] S. Kondaveeti, K. Prasad, A.K. Siddhanta, Carbohydr. Polym. 97 (2013) 165– 171. [11] H.H. Jeong, J.H. Lee, Y.M. Noh, Y.G. Kim, C.S. Lee, Macromol. Res. 21 (2013) 534–540. [12] R.S. Golightly, W.E. Doering, M.J. Natan, ACS Nano 3 (2009) 2859–2869. [13] J. Yoon, N. Choi, J. Ko, K. Kim, S. Lee, J. Choo, Biosens. Bioelectron. 47 (2013) 62– 67. [14] L. Wu, Z.Y. Wang, S.F. Zong, H. Chen, C.L. Wang, S.H. Xu, Y.P. Cui, Analyst 12 (2013) 3450–3456. [15] A. Huefner, W.L. Kuan, R.A. Barker, S. Mahajan, Nano Lett. 13 (2013) 2463– 2470. [16] S. Phadtare, A. Kumar, V.P. Vinod, C. Dash, D.V. Palaskar, M. Rao, P.G. Shukla, S. Sivaram, M. Sastry, Chem. Mater. 15 (2003) 1944–1949. [17] L.M. Liz-Marzan, Langmuir 22 (2006) 32–41. [18] H. Yoo, J.E. Millstone, S.Z. Li, J.W. Jang, W. Wei, J.S. Wu, G.C. Schatz, C.A. Mirkin, Nano Lett. 9 (2009) 3038–3041. [19] P. Aldeanueva-Potel, E. Faoucher, R.A. Alvarez-Puebla, L.M. Liz-Marzan, M. Brust, Anal. Chem. 81 (2009) 9233–9238. [20] R.B. Olivier, M. Jean-Francois, Anal. Chem. 83 (2011) 8057–8062. [21] L. Brus, Acc. Chem. Res. 41 (2008) 1742–1749. [22] J.P. Camden, J.A. Dieringer, Y. Wang, D.J. Masiello, L.D. Marks, G.C. Schatz, R.P. Van Duyne, J. Am. Chem. Soc. 130 (2008) 12616–12617. [23] G. Braun, I. Pavel, A.R. Morrill, D.S. Seferos, G.C. Bazan, N.O. Reich, M. Moskovits, J. Am. Chem. Soc. 129 (2007) 7760–7761. [24] M.J. Natan, Faraday Discuss. 132 (2006) 321–328. [25] E.C. Le Ru, E. Blackie, M. Meyer, P.G. Etchegoin, J. Phys. Chem. C 111 (2007) 13794–13803.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Preparation of gold nanoparticles–agarose gel composite and its application in SERS detection Xiaoyuan Ma a,b,c, Yu Xia a,c, Lili Ni a, Liangjing Song a, Zhouping Wang a,c,⇑ a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China The State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, PR China c Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, PR China b
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
Nanocomposites of agarose gel
embedded with gold nanoparticles were prepared. Dynamic hot-spots were generated from the agarose gel contraction loss of water. The nanocomposites were used successfully to detect Raman signal molecules. Recycling of the nanocomposites could be achieved with washing solution.
a r t i c l e
i n f o
Article history: Received 4 July 2013 Received in revised form 25 November 2013 Accepted 28 November 2013 Available online 7 December 2013 Keywords: Agarose gel Gold nanoparticles Nanocomposites SERS
a b s t r a c t Agarose gel/gold nanoparticles hybrid was prepared by adding gold nanoparticles to preformed agarose gel. Nanocomposite structures and properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and UV–Vis–NIR absorption spectroscopy. Based on the swelling–contraction characteristics of agarose gel and the adjustable localized surface plasmon resonance (LSPR) of the gold nanoparticles, the nanocomposites were used as surface enhanced Raman scattering (SERS) substrate to detect the Raman signal molecules (NBA, MBA, 1NAT). Results revealed that the porous structure of the agarose gel provided a good carrier for the enrichment of the gold nanoparticles. The gold nanoparticles dynamic hot-spot effect arising from the agarose gel contraction loss of water in the air greatly enhanced the Raman signal. Furthermore, the gel could be cleaned with washing solution and recycling could be achieved for Raman detection. Ó 2013 Elsevier B.V. All rights reserved.
Introduction In recent years, nanotechnology has received great advances in different research areas. Nanomaterials have been widely used in catalysis, biosensing detection and other fields due to its small size effect, high specific surface area and enhanced optical properties (quantum dot fluorescence effect, the quenching effect of gold material and surface-enhanced Raman properties, etc.) [1–3]. ⇑ Corresponding author. Tel./fax: +86 510 85917023. E-mail address: [email protected] (Z. Wang). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.11.111
Recently, a new functional material combined with nanostructures introduced into polymers has received widespread research interests. This kind of composite materials possess the advantages of both the nanostructures and the polymers which exhibit excellent performances. For example, with the combination of gold nanoparticles (GNPs) and TiO2, the photocatalysis properties could be greatly enhanced [4]. Up to now, nanocomposites have made great progress in sensors, microelectronic components, biochemical engineering (e.g. gene sequencing), chemical catalysts and other applications [5–8].
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Agarose is a kind of straight-chain polysaccharide extracted from seaweed or other vegetables. On heating to above 90 °C, the agarose powder is generally dissolved in water. When the temperature dropped to 35–40 °C, a good semi-solid gel will be formed. This is the main feature and foundation for a variety of usage. The gel gelation characteristics created by the presence of hydrogen bonds can be destroyed by any factor lead to the destruction of hydrogen bonds. The pore size of agarose could be changed by the concentration of agarose powder. It is because of this particular gelling properties combined with the significant stability, shrink and swell features as well as the easy absorption behavior, agarose gel has been widely used in food, pharmaceutical, chemical, textile, and other fields [9–11]. The surface-enhanced Raman scattering (SERS) phenomenon was discovered for more than 30 years of history. However, the spectrum is so weak to detect and the applications are limited. With the development of materials chemistry and instrument technology, especially the increased demand for trace detection, SERS has become one of the hot researches now. Substrate materials with a specific structure can effectively enhance the SERS signal. Here, nanomaterials with the high adjustable structural characteristics (size, shape, spacing, etc.) are excellent SERS substrates. Among them, the gold, silver nanomaterials due to their good optical absorption features are widely used in SERS studies. Owing to the high sensitivity and specificity of SERS technology, it has been widely applied in a majority of areas, including security, food safety monitoring, clinical diagnostics, trace analysis and biochemical analysis [12–15]. In this paper, the agarose gel was used as a template for the adsorption of prepared colloidal GNPs. The morphology and optical properties of the composite film were characterized. Combined with the shrink–swell properties of agarose gel and the optical absorption properties of GNPs, the composite film could be used as a good SERS substrate for the detection of Raman signal molecules, such as Niel blue A sulfate (NBA), 4-mercaptobenzoic acid (MBA), 1-naphthalenethiol (1NAT). The SERS spectra gradually increased due to the ‘‘hot spot effect’’ generated upon dehydration process when the agarose gel was exposed to the air as the network volume decreased and the GNPs got close to each other. Additionally, Raman molecules could be washed out when immersed in a cleaning solution, so that recycling could be achieved. Experimental details Materials Hydrogen tetrachloroaurate trihydrate (HAuCl44H2O), sodium borohydride (NaBH4), potassium carbonate (K2CO3), sodium citrate (C6H5Na3O7) were purchased from Shanghai Chemical Reagent Co., Ltd., China. Agarose was purchased from Sangon Biotech (Shanghai) Co., Ltd., NBA, MBA, 1NAT were purchased from Sigma–Aldrich. All the chemical reagents were of analytical grade or higher and used without further purification. Aqueous solutions used in the experiments were prepared using Milli-Q water from Milli-Q system (resistivity > 18 MX). All procedures were conducted at room temperature unless otherwise specified. Synthesis of GNPs GNPs (5 nm in diameter) were prepared by the reduction of AuCl4 with NaBH4 according to literature procedures [16]. Typically, an aqueous solution of HAuCl4 (200 mL, 36.4 mM) was first mixed with an aqueous solution of K2CO3 (1 mL, 0.2 M), and then reduced by the quick addition of a freshly prepared solution of NaBH4 (9 mL, 0.5 mg/mL1). The mixture turned wine red rapidly,
indicating the generation of GNPs. The obtained solution of GNPs was stored at 4 °C until use. Preparation of agarose gels Briefly, agarose hydrogels (1.5% w) were prepared by dissolving 225 mg of agarose powder in 15 mL of water and heated in microwave until complete dissolution to transparency and immediately poured into a ‘‘U’’ mold of 3 mm thickness and stored for at least 1 h at 4 °C. Then, the slab gel was cut into small films having a diameter of 1 cm and thickness of 3 mm using the hole puncher. Agarose gel film was immersed in ultrapure water and oscillated to remove the dispersed gel pieces. Synthesis of the gold nanoparticles–loaded agarose gel (GNPs– Agarose) Agarose gel film was immersed in a solution of GNPs. The GNPs could be spread to the gel membrane reticular formation to form GNPs–Agarose composite film. The corresponding UV–Vis–NIR absorption peak of the film was measured every once in a while and the peak position and intensity was recorded. SERS measurement NBA, MBA and 1NAT were selected as the Raman signal molecules (5 lM). Different concentrations of NBA were used to do the sensitivity detection ranging from 5 108 to 5 1010 M. The GNPs–Agarose composite film was immersed in a certain concentration of the solution in the NBA, MBA, 1NAT for the sufficient adsorption of probe molecules. Then the Raman absorption peak was measured in Raman spectroscopy with a Renishaw Invia Reflex system equipped with Peltier-cooled charge-coupled device (CCD) detectors and a Leica confocal microscope. Samples were excited with a 785 nm diode laser under linefocus mode and a grating of 1200 mm1 was used. The corresponding laser was focused onto the sample surface using a 50 long working distance objective. Spectra were collected in continuous mode with 10 s exposure time and accumulated for 2 times, the laser power was adjust to 0.05% which was about 0.06 mW. The changes of absorption peak were recorded over time. For reversible experiments, the GNPs– Agarose composite film was first immersed in analyte and the SERS spectra were recorded. Then, the film was washed by 1% sodium citrate solution and the SERS spectrum was again recorded. This procedure was repeated for at least 3 times. Results and discussion Preparation and characterization of GNPs–Agarose GNPs were prepared by the reduction of HAuCl4 using NaBH4 which called colloidal reduction chemical method. K2CO3 was added in the reaction system as a stabilizer and the protecting agent. Therefore, GNPs could be uniformly dispersed and stably present in the aqueous solution. Fig. 1(a and b) shows the TEM images and UV–Vis–NIR absorbance spectra of GNPs. GNPs were of spherical morphology with the diameter of 5 nm. And the plasmon peak was at 508 nm. The plasmon peak did not change apparently with time changes which illustrated that the gold colloid could be existed stably in solution. After the GNPs were attached to the agarose gel network, the morphological images and absorbance spectra were shown in Fig. 1(c and d). It can be seen that the GNPs were evenly distributed in the agarose gel film and were separated by a distance of gaps. The agarose gel network size and the mechanical strength can be adjusted by changing the
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Fig. 1. TEM images (a) and UV–Vis–NIR absorbance spectra (b) of GNPs. The morphological images (c) and absorbance spectra upon time from 1 h, 4 h, 8 h, 12 h, 15 h, 18 h, 20 h, 24 h respectively (d) of GNPs–Agarose gel.
concentration of the agarose gel. The agarose powder was dissolved in boiling water and then formed the porous structure under the cooling process. It could be used effectively as a natural porous template for the enrichment of the colloidal GNPs or other nanomaterials as a synthetic method for the composite structures combined with nanomaterials and organic materials. The absorbance spectra for GNPs–Agarose was slightly red shifted to 518 nm compared with the GNPs in solution. The LSPR absorbance spectra of the metal nanomaterials was affected by its morphology, particle size, degree of aggregation, environmental media refractive index and many other factors [17]. The red shift could be ascribed to the changes of the environmental media refractive index that the GNPs were first dispersed simply in water and then among the agarose gel network [18]. With the immersion time went on, more GNPs adsorbed to the gel membrane which made the absorbance intensity gradually increased. At the same time, some GNPs might stack to each other and resulted in the absorption peaks slightly red-shifted toward the long wavelength. By comparing the absorbance spectra of GNPs and the GNPs–Agarose composite gel in Fig. 1, we can see that about 90% GNPs were embedded in the network of agarose gel. So, about 1.97 1016 GNPs were loaded in the agarose gel film under the experimental conditions (suppose the diameter of GNPs was about 4 nm in average). SERS effect of GNPs–Agarose The agarose gel network loaded with high density of GNPs combined the swelling characteristic of the gel and the strong optical absorbance (localized surface plasmon resonance) of GNPs which made the composite materials good substrate in SERS detection. The enhanced SERS effect provided by these materials is related to the generation of a high density of hot spots when the gel
collapses upon dehydration. The consequent volume reduction of the materials drives the embedded gold colloidal nanoparticles close to each other, thus prompting the interaction between their respective electromagnetic fields and therefore further increasing the enhanced Raman signal. Fig. 2(a) shows the background SERS spectra of the dry, unloaded (with no Raman signal analyte) GNPs–Agarose and GNPs– Agarose with NBA molecules. Results presented a clean spectral window for the blank GNPs–Agarose gel. In contrast, upon immersion of the gel in an aqueous solution of NBA and subsequent airdrying, the SERS spectra showed well-defined bands with high intensity which are characteristics of NBA, including 592 cm1, 1141 cm1, 1351 cm1, 1429 cm1, 1492 cm1, 1544 cm1 and 1640 cm1. The uniformity of the entire surface was shown in Fig. 2(b) with eight Raman spectra at different detection point. It can be seen clearly that the Raman spectra remained almost the same at different detection area. The characteristic Raman shifts of NBA at 592 cm1 were used to calculate the relative standard deviation (RSD). A value of 5.44% for RSD was received which showed that the SERS enhancing ability of the GNPs–Agarose gel was homogeneous through the entire surface. It is an efficient and sensitive substrate for SERS detection. The dynamic hot spots induced SERS effect was demonstrated by using NBA as signal molecule during the dehydration process of the GNPs–Agarose gel. Fig. 3(a) depicts the substantial increase of the Raman signal. When the composite film loaded with Raman active molecule exposed to the air, the composite film dehydrated and shrinkaged. The reduced volume caused the GNPs closed to each other which resulted in a ‘‘dynamic hot spot effect’’ [19]. With time went by from 0 min to 120 min, the Raman peak appeared a 20-fold increase. This Raman signal enhancement effect may be caused by the following reasons. Firstly, in our experiment, when the agarose gel shrinked upon dehydration process, the imbedded
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Fig. 2. (a) SERS spectra of the dry GNPs–Agarose gel with (black line) and without (red line) NBA Raman molecule. (b) SERS spectra for uniformity of the entire GNPs–Agarose gel. (Eight random points were measured). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. (a) SERS spectra of GNPs–Agarose gel adsorbed with NBA Raman molecule upon dehydration on air from 0 min to 120 min. (b) SERS spectra obtained with different concentrations of NBA (a) 5 108 M, (b) 5 109 M, (c) 5 1010 M; (d) the spectrum measured while a non-SERS substrate was immersed in a solution with NBA concentration of 5 102 M.
GNPs got closer to each other which enhanced the aggregation extent and thereby generating the high electromagnetic fields. The enhanced LSPR features may cause the SERS intensity increasement [20]. And the distance between NBA molecules also decreased along with the dehydration process. So the number of NBA molecules at each unit laser spot increased which might also lead to SERS enhancement. Then, since the volume shrinkage of the composite gel causes the distance between GNPs decreases, the coupling between each other will have a ‘‘hot spot effect’’ [21– 23]. Because the surface enhancement factor (EF) is found to have many definitions in published literatures [24,25], in this manuscript, it is calculated by using the analytical chemistry point of view through the analytical EF (AEF) defined as [25]:
AEF ¼ ðISERS =C SERS Þ=ðIRS =C RS Þ where ISERS corresponds to the Raman intensity obtained for the SERS substrate under a certain concentration CSERS and IRS corresponds to the Raman intensity obtained under non-SERS conditions at an analyte concentration of CRS. According to this established definition, the AEF of the substrate was estimated by considering the 592 cm1 Raman shift, because it is the strongest peak of all bands in the spectra. When using CSERS = 5 109 M and CRS = 5 102 M and the intensities obtained from Fig. 3(b), we can obtain a value of about AEF = 107. It should be noted that the AEF is different when different Raman band or CSERS are chosen for estimation. But those AEF values are the same order of magnitude. Besides, we have compared the SERS substrate for GNPs–Agarose composite film and dispersed GNPs. Results (shown in Fig. S1) revealed that when the
agarose gel was wet before dehydration, the Raman intensity for GNPs–Agarose was slightly higher than GNPs themselves. While the agarose gel was collapsed upon dehydration, the Raman intensity was significantly enhanced. These data illustrated that the GNPs–Agarose film is superior than dispersed GNPs as SERS substrate. Fig. 3(b) shows the obtained SERS spectra when NBA solutions were added with different concentrations ranging from 5 108 to 5 1010 M. 5 1011 M NBA did not show any characteristic peaks, and thus the 5 1010 M concentration is taken as the limit of detection for the NBA molecule. The reversibility of the GNPs–Agarose gel was also studied for NBA and the results were shown in Fig. 4. The gel was first immersed in the solution containing the analyte, characterized by SERS, and then immersed in a washing aqueous solution of 1% sodium citrate, and the SERS spectra were measured again. This process was repeated three times to ensure the full reusability of this sensor platform. For NBA molecules, it is very likely that sodium and citrate ions compete for the retention sites of the analyte on the nanoparticles, thus displacing it because of their much higher concentration and thereby cleaning the sensor. When the analyte was again in contact with the gel, it was retained, giving rise to signals of similar intensity. As for other Raman signal molecules, for example, an anion (MBA) and a neutral molecular species (1NAT), the Raman spectra were obtained as shown in Fig. S2. When the GNPs–Agarose gel dehydrated in air, the Raman signal increased significantly. The same spectra changes could be explained as for Fig. 3. The
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of China (2012BAK08B01), the S&T Supporting Project of Jiangsu (BE2011621, BE2012614), funding from the State Key Laboratory of Bioelectronics of Southeast University. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.11.111. References
Fig. 4. Reversible SERS spectra of GNPs–Agarose gel after adsorbed with NBA Raman molecule (dry film) and washed with sodium citrate solution for three times. (The upper line is the gel with analyte and the lower line is the gel washed with sodium citrate).
reversibility experimental results (Fig. S3) revealed that the composite films could also be reused for anion and neutral molecules detection. Conclusion The agarose gel-based porous structures were used as template to adsorb prepared GNPs. The morphological and optical properties of the composite GNPs–Agarose gel hybrid were characterized and analyzed using TEM, SEM and UV–Vis–NIR. When the composite film was exposed to the air, the agarose gel dehydrated and shrinked. The GNPs got close to each other and the hot-spot effect generated. Results revealed that this composite film could be used as an effective SERS substrate for the detection of Raman signal molecules (NBA, MBA, 1NAT). The SERS signal gradually increased upon the dehydration process due to the dynamic hot-spot effect. Additionally, this composite material could be cleaned by washing solution and be reused when immersed in Raman molecules. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grant 21375049), National S&T Support Program
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