Au nanosheets

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 142 (2015) 50–54

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A sensitive SERS substrate based on Au/TiO2/Au nanosheets Li Jiang a, Xiu Liang a, Tingting You a, Penggang Yin a,⇑, Hua Wang a, Lin Guo a,⇑, Shihe Yang a,b,⇑ a b

Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, School of Chemistry and Environment, Beihang University, Beijing 100191, China Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

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

 An easy-prepared Au/TiO2/Au

nanosheet are fabricated as SERS substrate. 7  Stronger SERS signal (EF is 10 ) is found as compared to the Au/Ti system.  Influence factors as concentration and deposition time were discussed.  The substrates are with good homogeneity (RSD <10% by Raman mapping).  The substrates can be applied in label-free detection of adenine.

a r t i c l e

i n f o

Article history: Received 17 July 2014 Received in revised form 22 December 2014 Accepted 14 January 2015 Available online 28 January 2015 Keywords: SERS TiO2 AuNPs PMBA Adenine

a b s t r a c t Sensitive SERS substrates based on Au/TiO2/Au nanosheet have been prepared by physically sputtering Au nanoparticles onto fabricated TiO2 nanosheets. The Au/TiO2/Au nanosheets show much stronger SERS signal as compared to normal Au/Ti substrates by increasing surface area and effectively inducing plasmonic coupling between adjoining Au nanoparticles. In addition, influence factors such as concentration of probe solution and deposition time of gold nanoparticles were discussed. This study provides an easyprepared and label-free substrate for the detection of biomolecule. Ó 2015 Elsevier B.V. All rights reserved.

Introduction Since its discovery in 1974, surface-enhanced Raman spectroscopy (SERS) has drawn increasing attentions due to its capability in trace-level molecule detection [1–3]. The potential applications are in various fields, including chemical analysis, biomolecule detection and structure dynamics elucidation of molecules transformation [4–6]. Various approaches have been ⇑ Corresponding authors at: 37 Xueyuan Road, Haidian District, Beijing 100191, China. Tel.: +86 10 82338987; fax: +86 10 82338987 (P.G. Yin). E-mail addresses: [email protected] (P. Yin), [email protected] (L. Guo), [email protected] (S. Yang). http://dx.doi.org/10.1016/j.saa.2015.01.040 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

employed to fabricate efficient and reproducible SERS substrates, such as nanoparticles, core–shell particles, thin films and nanowires, etc. [7,8] Recently, scaffold-based hybrid nanostructure for SERS substrates were fabricated with template method [9–11]. The support materials were decorated with nanoparticles that induced ‘‘hot spots’’ in the gaps and provided several orders of magnitude enhancement [12]. Various materials have been used as support component such as nanoporous GaN [13,14], Si [15] and ZnO [16,17]. Being an important wide gap semiconductor with particular optical and chemical properties [18,19], TiO2 is also a good candidates for support material [20,21]. Several research groups decorated Au or Ag nanomaterials on the surface of TiO2 nanotubes, nanorods or nanospheres [22–24].

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In this work, we fabricated an easy-prepared SERS substrate base on Au/TiO2/Au nanosheets. The TiO2 nanosheets were directly fabricated on Ti foil and decorated by Au nanoparticles using physical sputtering system. The inducing of perpendicular grown TiO2 between planar Ti substrate and decorated Au nanoparticles could provide larger surface area for more metal NPs and molecules, and enable coupling along different directions in three dimensions. 4-Mercaptobenzoic acid (4-MBA) was employed as probe molecules for SERS characterizations. Influence factors such as solution concentration of probe molecules and deposition times of metal nanoparticles were discussed. A direct detection on adenine [25– 27] molecules was carried out after a spot by spot Raman mapping experiment that evinced the sensitivity and homogeneity of the substrate in large area. Experiment section Chemical reagents and sample preparation 4-Mercaptobenzoic acid (4-MBA, 99%) was purchased from Acros Organics and used without further purification. Adenine was purchased from Sigma–Aldrich. High-purity water (Millipore) was used throughout. TiO2 nanosheets were fabricated by hydrothermal synthesis according to previous work [28,29]. Briefly, a cleaned titanium foil was put in aqueous ammonia solution and kept at 100 °C for several days. Then the sheets were sintered at 500 °C in air to obtain TiO2 films. Au sputtering system (JMB-3000VA, GUANGTAI Electric Ltd., China) was used to decorate gold nanoparticles and form Au/TiO2/Au structure based on nanosheets substrates. SERS samples were prepared by immersing Au/TiO2/Au nanosheets into 4-MBA or adenine solution for five hours before being rinsed with ethanol several times to remove the free molecules. Sample characterization The morphologies of the samples were carried out by field-emission gun scanning electron microscope (SEM, Hitachi S-4800, 5 KV). Raman spectra were studied with a confocal 180° backscattering geometry micro-Raman spectrometer (Jobin Yvon, HR800). The 647 nm from a Ar–Kr ions laser was used as exciting source. The scatter light was analyzed with a Dilor XY triple spectrometer and a liquid-nitrogen-cooled CCD multi-channel detector. The laser power on the surface of samples was typically about 5 mW. The accumulation time was 10 s and 5 times for all the spectra. 520 cm1 peak of silicon wafer was used to calibration the spectrograph.

Fig. 1. SEM images of neat TiO2 nanosheets grown on Ti films (a, b) and the asprepared Au/TiO2/Au nanosheets (c, d) at low (a, c) and high (b, d) magnifications.

SERS properties of Au/TiO2/Au nanosheets Scaffold-based hybrid nanostructure offers high-density ‘‘hot spots’’ as well as larger surface area for probe molecules, making it a promising candidate of SERS substrates [24]. In order to investigate the SERS activity, as-prepared Au/TiO2/Au nanosheets were modified by 4-MBA molecule (105 M) before being subjected to Raman measurement. Normal Raman spectrum of 0.2 M 4-MBA ethanol solution was shown in Fig. 2(a) for comparison. Distinct Raman bands were obtained while much reduced background

Results and discussion Characterization of the Au/TiO2/Au nanosheets Morphology of neat TiO2 nanosheets was characterized by SEM in Fig. 1(a) and (b). The as-prepared TiO2 nanosheets were perpendicularly grown on titanium foil with an average thickness of about 5 nm. The TEM and HRTEM image of the TiO2 nanosheets in previous work further revealed the anatase phase of TiO2 in nanosheets [29]. As shown in Fig. 1(c) and (d), Au nanoparticles were deposited on the surface of TiO2 nanosheets by electron beam evaporation. Size distribution data was measured from amplified SEM images at high magnifications (Fig. 1(d)) as shown in Fig. S1. Average diameter and size error was estimated to be 15 ± 9 nm. Energy dispersive X-ray analysis (EDAX) results in Fig. S2 (see Electronic Supplementary Material) confirm the existence of Au nanoparticles.

Fig. 2. (a) Normal Raman spectra of 0.2 M 4-MBA ethanol solution. (b) Normalized SERS spectra of 4-MBA (105 M solution) adsorbed on the surface of Ti/Au foil. (c) Normalized SERS spectra of 4-MBA (105 M solution) adsorbed on the surface of Au/ TiO2/Au nanosheets. (d) SEM image of as-prepared Au/TiO2/Au nanosheets at high magnifications. All normal and SERS spectra have been normalized with the Raman intensity of band at ca. 1585 cm1 obtained from sample with 4-MBA (105 M solution) adsorbed on the surface of Au/TiO2/Au nanosheets (c).

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signals could be observed as the physical sputtering method helped to avoid external chemical capping agent. In SERS spectrum (Fig. 2(c)), the in plane breathing mode coupled mCS mode shifted from 1100 to 1074 cm1, indicating that the 4-MBA molecules bind to gold surfaces through thiol groups. Band at 1585 cm1 can be attributed to m12 (a1) aromatic ring breathing mode. The weak peaks at 1421 and 850 cm1 arose from COO stretching and bending modes, while the bands at 1140 (m15, b2) and 1179 cm1 (m9, a1) were C–H deformation modes [30,31]. Similar Raman experiment was also performed on Au/Ti system, which was prepared by performing same Au sputtering process on the surface of neat Ti foil (The corresponding SEM images were shown in Fig. S3). As can be seen in Fig. 2(b), an obviously lower signal was obtained from molecules adsorbed on Ti/Au surface as compared to the Au/ TiO2/Au nanosheets. Since deposition time was fixed as 8 s for both substrates, the amounts of gold nanoparticles should be less varied. This fact indicates that, apart from larger surface area for metal nanoparticles and probe adsorbates, the inducing of TiO2 nanosheets also provide a 3-dimensional space for decorated gold nanoparticles compared to planar surface of Ti, which might benefic the SERS performance. The bands at 1585 and 1074 cm1 of 4-MBA are chosen to estimate the magnitude of the SERS enhancement factor (EF), based on the method in reference: [32].

EF ¼ ðISERS =Nads Þ=ðIbulk =Nbulk Þ

ð1Þ

where ISERS and Ibulk are the intensities of the same band in SERS spectra and normal Raman spectra of bulk probe molecules; Nbulk and Nads are the number of 4-MBA molecules under the laser illumination for the bulk and SERS experiments, respectively. Nbulk is obtained by Ahq/m, where A, h, q and m are the area of laser spot (1.4 lm2), the penetration depth of the focus laser (10 lm), the density (1.5 g cm3) and the molecule weight (154.19) of 4-MBA. Nads is given by multiplying the surface densities of 4-MBA (0.5 mol cm2) [32] and the laser spot area. By substituting values into Eq. (1), EF for the SERS of 4-MBA adsorbed on Au/TiO2/ Au nanosheets substrate was estimated to be about 107. Raman spectra of Au/TiO2/Au nanosheets modified by 4-MBA solution with different concentrations have been investigated as shown in Fig. 3. With the concentration of adsorbates varied from 10 lM to 1 nM,

Fig. 3. Normalized SERS spectra of 4-MBA adsorbed Au/TiO2/Au nanosheets at variable 4-MBA solutions concentrations: (a) 105 M, (b) 106 M, (c) 107 M, (d) 108 M and (e) 109 M. All SERS spectra have been normalized with the Raman intensity of band at ca. 1586 cm1 obtained from sample with 4-MBA concentration of 105 M (a).

the intensities of typical spectral bands decreased and the Raman signals are still clearly observed when the solution concentration is as low as 1 nM. To explore the relationship between nanoparticles amount and SERS intensity, Au/TiO2/Au nanosheets were prepared with different Au deposition time. According to SEM images in Fig. S4 (see Electronic Supplementary Material), the density of Au nanoparticles was obviously increased when the deposition time became longer. Gold nanoparticles are with a size of 5 nm in samples under 2 s deposition process. The nanoparticles started to aggregate when the deposition time was 4 s, while the aggregations tend to be more resemble to island on sample using a deposition time of 8 s. This change in density increased the average size up to 11 nm as shown in Fig. S1, leading to larger surface coverage of gold and thus better performance during SERS measurements. Besides strong enhancement arising from ‘‘hot spots’’ in the gap from island structure might play a role as well [14]. Fig. 4 shows the Raman spectra of 4-MBA molecules adsorbed on the surface of Au/TiO2/Au nanosheets with different deposition time: 2 s (a), 4 s (b) and 8 s (c) with fixed adsorbate concentration and experiment conditions. Homogeneity of spectral signal through an area is of great importance when considering the practical application using SERS substrates. As shown in Fig. 5, we performed a mapping measurement via spot to spot Raman spectra on a 20  20 lm Au/TiO2/Au area with a step size of 1 lm to evaluate the homogeneity of SERS signals. SERS spectra of 4-MBA obtained from different spots are with good stability, since the nanosheets substrate help for avoiding the ring-like structure caused when liquid evaporate and leave analyte molecules on the substrate [33]. The relative standard deviation (RSD) of the Raman intensity was calculated to be 8.5% for 1585 cm1 and 8.6% for 1074 cm1, indicating the uniformity of SERS substrate in large area.

SERS detection on adenine molecules In recent years, SERS has been developed for rapid detection and quantification of nucleic acids with multiple cellular functions [6,25–28]. Among these biological molecules, adenine is of special interest for its widespread existence in metabolome and easy chemisorptions to Au surface. Recent studies have revealed that

Fig. 4. Normalized SERS spectra of 4-MBA molecules adsorbed on Au/TiO2/Au nanosheets with different Au deposition times: 2 s (a), 4 s (b) and 8 s (c). All SERS spectra have been normalized with the Raman intensity of band at ca. 1585 cm1 obtained from sample with Au deposition time of 8 s (c).

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Fig. 5. (Top) SERS spectra of 4-MBA on several spots of the Au/TiO2/Au nanosheets; (a and b) Raman intensity maps at 1585 and 1074 cm1 of 4-MBA molecules on a 20  20 lm surface area of the Au/TiO2/Au nanosheets; (c and d) The corresponding intensity distributions at 1585 and 1074 cm1 of 4-MBA molecules on a 20  20 lm surface area of the Au/TiO2/Au nanosheets.

the adenine ring-breathing band greatly dominates the SERS spectra of DNA or RNA biomolecules, which makes the analyte even more important in the field of biomolecule detection [6,27]. SERS studies of adenine have been employed on several substrates such as silver and gold electrodes, gold colloids [34,35]. In this work, Au/ TiO2/Au nanosheets substrate were directly used for adenine solutions of different concentrations. As shown in Fig. 6, the SERS spectra of adenine on the nanosheets are dominated by the adenine in-plane ring breathing mode at 737 cm1 [34,35]. The band could still be recognized clearly while the sample concentration is as low as 107 mol L1. As the local electromagnetic field enhancement caused by localized surface plasmon resonance (LSPR) plays an important role in SERS, three dimensions TiO2 nanosheets with gold nanoparticles depositing on the surface bring in various types of ‘‘hot spots’’ or ‘‘nanogap’’ [36–37] formed in the aggregation of gold nanoparticles. Electromagnetic coupling effect results in far-field phenomenon such as hybridizations in optical modes as well as spatially confined near-field electromagnetic enhancement in gap

region. In the hybrid structure, TiO2 nanosheets act as supporter for ‘‘hot spots’’ formed by gold nanoparticles, while in some cases the semiconductor material was also reported to influence the optical properties by introducing charge-transfer or interface effect [38,39]. The prepared TiO2 nanosheets provided 3-dimensional space for decorated gold nanoparticles compared to planar surface of Ti, inducing more possibilities of hot spots in nanostructure [40]. For substrate with longer deposition time, the aggregation of island-like nanoparticles would exhibit a maximum enhancement in the gap region as illustrated in reference [41].

Conclusions In summary, a facile method was used to fabricate Au/TiO2/Au nanosheets, which were further used as SERS substrates to achieve high sensitivity and reproducibility. Compared to Au/Ti substrates system, Au/TiO2/Au nanosheets show much stronger SERS signal by effectively inducing plasmonic coupling resonance between

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Fig. 6. Normalized SERS spectra of adenine adsorbed on Au/TiO2/Au nanosheet at variable adenine solutions concentrations: (a) 104 M, (b) 105 M, (c) 106 M and (d) 107 M.

located Au nanoparticles as well as providing larger surface area for nanoparticles and molecules. The substrates have also exhibited good homogeneity during Raman mapping experiments. Influence factors such as concentration of probe solution and deposition time of gold nanoparticles were discussed to investigate the effect of surface coverage. Finally, the nanosheet-based hybrid substrates were directly employed for measurement of adenine molecules and proved to be available for label-free detection of biomolecules as well. Acknowledgments This work was supported by the National Natural Science Foundation of China (51002007 and 51272013) and the Innovation Foundation of BUAA for PhD Graduates. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2015.01.040. References [1] M. Fleischmann, P.J. Hendra, A.J. McQuillan, Chem. Phys. Lett. 26 (1974) 163– 166. [2] D.L. Jeanmaire, R.P. Van Duyne, Surf. Raman Spectroelectrochem. 84 (1977) 1–20.

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