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Graphene isolated Au nanoparticle arrays with high reproducibility for high-performance surface-enhanced Raman scattering
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Graphene isolated Au nanoparticle arrays with high reproducibility for high-performance surface-enhanced Raman scattering Shicai Xu a,∗ , Shouzhen Jiang b , Jihua Wang a , Jie Wei c , Weiwei Yue b , Yong Ma b a College of Physics and Electronic Information, Shandong Provincial Key Laboratory of Functional Macromolecular Biophysics, Dezhou University, Dezhou 253023, China b College of Physics and Electronics, Shandong Normal University, Jinan 250014, China c Department of Neurology, Dezhou People’s Hospital, Dezhou 253014, China
a r t i c l e
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Article history: Received 17 June 2015 Received in revised form 2 August 2015 Accepted 5 August 2015 Available online xxx Keywords: Graphene Au nanoparticle arrays Nanostructure SERS Adenosine
a b s t r a c t An efficient surface enhanced Raman scattering (SERS) substrate has been developed based on highly ordered arrays of graphene–isolated Au nanoparticle (G/AuNP). By combining the electromagnetic enhancement activity of AuNP arrays and unique physical/chemical properties of graphene, the G/AuNP arrays shows high performance in terms of sensitivity, signal-to-noise ratio and reproducibility. The average enhancement factor obtained from the G/AuNP arrays for rhodamine 6G probe molecules is over 107 . The maximum deviations of SERS intensities from 20 positions of a same SERS substrate are in the range of 4.74% to 6.89% and from 20 different substrates in various batches are in the range of 5.67% to 8.37%, depending on different vibration modes. As a practical application of this SERS system, we detect the adenosine concentration in human serum. The detection results show a good linear correlation between SERS intensity and adenosine concentration within the range of 2 to 250 nM. This work may open up new opportunities in developing the applications of SERS in biomedical diagnostics, biological sensing and other biotechnology. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Surface-enhanced Raman scattering spectroscopy is a powerful technique for ultrasensitive and selective detection in biology and chemistry [1–3]. Although Raman spectroscopy is limited by low sensitivity, SERS can provide signal intensity of the molecules enhanced by orders of magnitude on the proper substrates. Though the detailed physical mechanism of SERS is still under debate, it has been widely accepted that SERS enhancement results from a combination of an electromagnetic mechanism (EM) and a chemical mechanism (CM) [4]. The EM originates from the dramatic increase in the local electromagnetic field due to a light-excited surface plasmon resonance, which can boost the pristine Raman signal by 108 times or more [5,6]. Yet for CM, it is caused by charge transfer between the target molecules and the substrate, usually has a minor enhancement factor of 10–102 [7]. Both experimental and theoretical results indicate that the EM enhancement of SERS is from metal nanoparticles (NPs) and the enhancement factor depends on the gap between adjacent NPs [8–10]. A great deal of research
∗ Corresponding author. Tel.: +86 5348982081. E-mail address: [email protected] (S. Xu).
effort in SERS community has been focused on the fabrication of controlled and reproducible metallic nanostructures incorporating as much as possible the hot spots, including the colloidal clusters of noble metallic NPs [11–13], surface-roughened noble metals [14], and well-defined top-down fabricated metal architectures [15,16], such as Ag nanoclusters on ZnO nanodome arrays [17] well-ordered Au-nanorod arrays [18], Au nanorods coupled with Ag nanoparticles [19]. During the past four decades, great efforts and ingenuity have gone toward creating a more desirable SERS substrate, resulting in extraordinary sensitivity ultrasensitive [20,21], or even at the single-molecule level [22–24]. Yet SERS has not entered the widespread real world application stage. The main problem is that the general SERS substrates based on Ag and Au, either in the form of dispersed nanoparticles or ordered nanoparticles, nanowires, and nanorod arrays are difficult to release high reproducible SERS signals for facing many challenges: first, it is required to have a large-area SERS substrate with regularly arranged nanostructures containing well-controlled narrow gaps that can generate the homogeneous hot spots; second, it is also required to capture the interested molecules effectively and homogenously on the surface of the SERS substrate; third, the large fluctuations of SERS signal need to be minimized. The sources of fluctuations include dynamic interaction of the target molecules with the local environment of plasmonic metal [25,26], the change of the adsorption
http://dx.doi.org/10.1016/j.snb.2015.08.009 0925-4005/© 2015 Elsevier B.V. All rights reserved.
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sites caused by the restructuring of the substrate surface [27], the drift of analyte molecules on substrate surface caused by excitation laser [14]. These challenges cause low SERS signal even in the most sophisticated measurements. Because of the poor reproducibility, the practicality of the SERS has always been questioned by the non-SERS community. While people have strived to deal with the first challenge to create reproducible structures by fabricating well-defined SERS active substrates [8,15], the latter problems may be equally important but ignored to some extent. Recently, a thin and pinhole free layer of SiO2 or Al2 O3 as an inert shell was employed in the SERS substrates to isolate metal nanostructures from their surroundings [28]. This strategy shows one overwhelmingly attractive feature that the SERS signal derives only from the target analytes and spurious signals induced by the metal is locked by inert shell. Fabrication of SERS substrates with an ultrathin passivated surface at a lowest loss of electromagnetic enhancement activity is the key to shell-isolated SERS. The main challenge is to get a pinhole-free coating layer with a very small thickness. Graphene, a 2D atomic crystal with densely packed carbon atoms in a honeycomb crystal lattice [29], is well-known for its unique electrical performance and the amazing applications in nanoscale electronics [30]. As well as the wide use in electronics field, the atomic thickness and seamless structure of graphene also makes it a natural candidate material for shell-isolated SERS [31]. The ultrathin graphene layer can provide effective transfer of the electromagnetic field from the metal core through the shell to the probe molecule, at the same time the stable and pinhole-free shell can protect the inner metal core and prevent the direct metal–molecule contact. Besides the isolation function like SiO2 or Al2 O3 shell, graphene layer as a new kind of shell also has many other functions. Typical functions include surface adsorption [32], additional chemical enhancement and fluorescence quenching [33]. To take advantage of these functions of graphene, many efforts have been done to fabricate graphene-isolated metallic nanostructure for SERS. Xu et al. prepared a graphene–veiled gold substrate with a passivated surface for SERS [14]. Xie’s group fabricated a hybrid SERS-active platform consisting of a single layer graphene covering a quasiperiodic Au nanopyramid that enabled single molecule detection for rhodamine 6G (R6G) [31]. Zhu’s group has shown that graphene–gold nanoparticle hybrid films is a promising material for shell isolated SERS [34]. Li et al. fabricated monolayer graphenecovered Ag nanoparticles and demonstrated that the graphene layer can obviously suppress the oxidation of Ag nanoparticles [35]. These works provided facile methods to use graphene to wrap metal nanoparticles and more stable SERS signals were obtained. However, in these studies, either for the colloidal clusters of noble metallic NPs [34,35] or surface-roughened noble metals [14], the hot spots are randomly distributed on the SERS-active substrates. Although the graphene as a kind of additive has been employed in SERS substrates, high reproducible SERS signals are still difficult to achieve due to the spatial inhomogeneity of these plasmonic substrates. Thus it is necessary to design a kind of SERS substrate to obtain high reproducible SERS signals by combining the high ordered structure of plasmonic metal nano arrays with the multifunctions of graphene. Herein, we demonstrate that clean and high reproducible SERS signals were obtained by combining large-area AuNP arrays with monolayer graphene. The G/AuNP arrays boost a high density of hot spots with average SERS enhancement factor over 107 for R6G detection. The added graphene layer enables SERS analysis with more well-defined molecular interactions and thus improves the reproducibility of SERS signals. The maximum fluctuations from 20 positions of a same SERS substrate are less than 7% and from 20 different substrates in various batches are less than 9%. The high reproducibility of the enhanced Raman signals can be attributed to the well-ordered morphology of AuNP arrays and
multi-functions of graphene layer including metal–molecule isolation, surface adsorption, and fluorescence quenching. On the basis of the AuNP array substrates, the adenosine SERS signals with high selectivity and reproducibility were detected in human serum. Adenosine is the metabolite of adenine nucleotides, which has been regarded as one of the major neuromodulators. As the core molecule of ATP and of nucleic acids, adenosine forms a unique link among cell energy, gene regulation, and neuronal excitability [36]. The successful detection of adenosine in human serum indicated that the G/AuNP array substrates have great potential for detecting other analytes in real biological systems. 2. Experimental 2.1. Materials and instruments R6G and adenosine were purchased from Aladdin Industrial Corp. Ltd (Shanghai, China) as test samples. Human serum was provided by Department of Internal Medicine of Dezhou People’s Hospital for the feasibility test of the G/AuNP array substrates. The morphologies of AuNP arrays were characterized by scanning electron microscopy (SEM, ZEISS, SUPRATM-55). The transmission electron microscopy (TEM) images were obtained by a transmission electron microscope system (JEOL JEM-2100) with X-ray energy dispersion analysis equipment (EDS, JEOL Company). For TEM analysis, the quartz-supported AuNP arrays was immersed in 35% HF solution for 2 h to free individual particles from the substrates. The SERS experiments and the graphene characterization were carried out in a Raman spectrometer (Horiba HR-800) with laser excitation at 532 nm. 2.2. Preparation of AuNP arrays The AuNP arrays were fabricated by electron beam evaporation using anodic alumimum oxide (AAO) as template. The throughpore AAO templates were prepared via a two-step anodization of pure aluminum foils (99.999%). The first anodization was performed under constant potentials of 40 V in 0.3 M oxalic acid at 20 ◦ C for 7 h. Secondary anodization was carried out under constant potentials of 40 V in 0.4 M phosphoric acid at 20 ◦ C for 10 min. After the barrier layer was removed, the pores were widened via wet chemical etching in 5 wt% phosphoric acid at 30 ◦ C for 40 min. By using through-pore AAO as template, a gold layer of about 100 nm was evaporated onto the quartz substrates. Finally, the AAO template was completely removed by immersing in 5% phosphoric acid to expose the AuNP arrays on the quartz substrates. 2.3. Preparation of monolayer graphene The large-area graphene was synthesized on a piece of copper foils (25 m thick) in a quartz tube at 1050 ◦ C by chemical vapor deposition (CVD) as reported in detail in our previously study [37]. First, the copper foils were annealed for 10 min with flowing hydrogen of 15 sccm at 90 Pa to increase the copper grain size [38]. Then, the gas mixture of methane (10 sccm) and hydrogen (65 sccm) was introduced into quartz tube at 200 Pa for 20 min to grow graphene. Finally, the samples were rapidly cooled to room temperature with flowing hydrogen at rate of 65 sccm. 2.4. Preparation of G/AuNP arrays The preparation processes of G/AuNP arrays are summarized in Fig. 1. By using CVD method, large-area graphene was grown on a piece of Cu foil (Fig. 1a and b). Polymethylmethacrylate (PMMA) was spincoated on one side of the foil and the Cu foil was subsequently etched away using 0.1 M aqueous FeCl3 solution for 20 min
Please cite this article in press as: S. Xu, et al., Graphene isolated Au nanoparticle arrays with high reproducibility for high-performance surface-enhanced Raman scattering, Sens. Actuators B: Chem. (2015), http://dx.doi.org/10.1016/j.snb.2015.08.009
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Fig. 1. Preparation processes of G/AuNP array substrates.
(Fig. 1c and d). Then the graphene/PMMA film was washed with HCl/H2 O (1:10) and deionized water for three times and then transferred onto the quartz-supported AuNP arrays (Fig. 1e). To avoid the breakage of the graphene, we employed a small amount of liquid PMMA to introduce a second PMMA coating on the precoated PMMA/graphene/AuNP arrays. The redissolution of the precoated PMMA tends to mechanically relax the underlying graphene [39], leading to a better contact with the surface morphology of the underlying AuNP arrays (Fig. 1f and g). Then the PMMA layer completely removed by being dissolved in dichloromethane (DCM) at 50 ◦ C within several minutes (Fig. 1h). Finally, the substrate was rinsed with isopropyl alcohol and the graphene isolated AuNP array substrates were formed (Fig. 1i).
nanodots after removal of the AAO template. The hexagonally arranged Au particles are almost identical in size and topology. The average diameter of the particles is ∼90 nm, and the distance between two neighboring particles is ∼60 nm. The period of the nanoparticle array is ∼150 nm, which is consistent with the period of the AAO template. Fig. 2c shows the TEM image of Au particles that have been completely liberated from the quartz substrates. The image shows that the Au particles are spherical and the average diameter of the particles is ∼90 nm, in good agreement with the result of the SEM images. Fig. 2d shows EDS spectrum from the Au particles. All the peaks are associated to Au element, demonstrating a high purity of Au particles. 3.2. Characterization of G/AuNP array substrates
2.5. SERS experiments SERS experiments were carried out using a Raman system (Horiba HR-800) with laser excitation at 532 nm. The excitation laser spot was about 1 m, and the effective power of the laser source was kept at 0.05 mW for R6G molecules, and 0.25 mW for adenosine molecules. The system was connected to a microscope, and the laser light was coupled through an objective lens of 20×, which was used for exciting the samples as well as collecting the Raman signals. Prior to each Raman experiment, calibration of the instrument was done with the Raman signal from a silicon standard centered at 520 cm−1 . SERS substrates were incubated for 3 h in different analyte solutions and then were placed into a vacuum drying oven to evaporate any moisture. The substrates were taken out and fixed onto the glass slide for SERS measurements. The SERS measurements were performed from at least 8 random locations that were more than 3 mm apart with an accumulation time of 10 s. If there is no special instruction, the mentioned Raman spectra are expressed in terms of average spectra. 3. Results and discussion 3.1. Characterization of AuNP arrays Fig. 2a shows the SEM image of a typical AAO membrane. The typical area of the nanohole arrays is defect-free and these nanoholes with uniform size are hexagonally arranged in high regularity. The mean diameter and the interval of the holes are ∼100 nm and ∼50 nm, respectively. The AuNP arrays were fabricated through a standard electron beam evaporation technique using the AAO template. Fig. 2b shows a representative SEM image of Au
Fig. 3a shows the SEM top- and cross-section (inset) images of the hybrid structure of G/AuNP array substrates. Graphene deforms itself to conform to the AuNP geometry and only a few of breakages is observed as marked by the white arrows. The cross-section image shows a clear lateral-view of G/AuNP arrays, consistent with the schematic structure of G/AuNP arrays (Fig. 1). As both SEM top- and cross-section images cannot give direct evidence of graphene, a conventional Raman measurement was performed on these hybrids to demonstrate the existence of graphene. As shown in Fig. 3b, the D, G and 2D peaks of graphene are clearly observed at ∼1360, ∼1580 and ∼2695 cm−1 , respectively. These peaks can be regarded as the fingerprint of graphene [40,41]. The Raman spectrum shows typical features of monolayer graphene: the intensity ratio of I(2D) /I(G) ≥ 2 and a single Lorentzian 2D peak with a full width at half maximum (FWHM) of ∼35 cm−1 [42]. Here, the defectrelated D peak is very weak, indicative of high quality of graphene. These results demonstrate that the graphene film with a monolayer structure is actually transferred on AuNP arrays. To evaluate the uniformity of graphene, Raman mappings of graphene on AuNP arrays were implemented over 60 m × 60 m area as shown in Fig. S1 in the Supporting information. From the color scale in Fig. S1a, it can be seen that the intensity ratios of 2D-peak to G-peak (I2D /IG ) show a very small fluctuation from 2.6 to 2.8, indicating that the monolayer graphene have a very uniform structure in a large area. Disorder-induced D-peaks were also detected with the intensity ratio of the D-peak to G-peak (ID /IG ). The Raman ID /IG ratio was extensively used to estimate the quality of graphene and the degree of disorder. As shown in Fig. S1b, the intensity ratios of ID /IG are less than 0.07, indicating that the employed graphene has little defects even in a large area.
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Fig. 2. (a) SEM image of through-pore AAO membrane. (b) SEM image of AuNP arrays. (c) TEM image of Au particles liberated from quartz substrate. (d) EDS spectrum of the Au particles.
3.3. SERS activity of G/AuNP arrays To verify the SERS activity of G/AuNP array substrates, we compared the performance of G/AuNP array substrates with that of AuNP array substrates. By maintaining the same measurement conditions, the R6G aqueous solutions with varied concentrations were used as the probe molecule. Fig. 4a shows the SERS spectra of R6G molecules from AuNP array substrates with various concentrations of 10−7 , 10−8 , 10−9 , 10−10 M, respectively. The Raman peaks at 612, 773, 1185, 1311, 1362, 1506, 1570 and 1647 cm−1 are in good agreement with previous reports for R6G [43,44]. Beside of characteristic peaks of R6G, some additional weak peaks are also observed as marked by red arrows. These additional peaks are difficult to be assigned precisely, which can be attributed to the various possible interactions between AuNPs and the R6G molecules, including charge transfer between the metal and molecules, photo-induced damage and metal-catalyzed side reactions, etc. [27]. The quality of SERS signals in terms of sensitivity and signal-to-noise ratio was improved on the G/AuNP arrays. As shown in Fig. 4b, the additional weak peaks and fluorescence background in the SERS spectra are obviously reduced and the characteristic signals of R6G are still visible even at 10−11 M. Because of the additional graphene layer, the R6G molecules are isolated from metal AuNP arrays and the spurious SERS signals from various possible metal–molecule interactions are thus reduced. Though a few of graphene breakages is observed on the G/AuNP arrays, the additional graphene layer still shows great isolation effect. That’s probably because most of the hot spot is located in between two consecutive AuNPs, whereas few graphene breakages exist in this region as shown in Fig. 3a. The reduction of fluorescence background can be explained by the fluorescence quenching effect of graphene. This effect is basically due to a resonance energy transfer process from dye molecules to graphene as reported from Swathi et al. [45]. The G peaks of
graphene are also clearly observed, which are nearly independent of the concentrations of R6G. The SERS enhancement factors (EF) for R6G on the G/AuNP arrays were calculated according to the standard equation [46–48]: EF =
ISERS /NSERS IRS /NRS
(1)
where ISERS and IRS , respectively, represent the peak intensities of the SERS spectra and the normal Raman spectra, NSERS and NRS are, respectively, the numbers of molecules on the substrates within the laser spot. In the experiments, a certain volume VSERS and concentration CSERS R6G aqueous solution was dispersed to an area of SSERS on the SERS-active substrates. For normal Raman experiment, a certain volume VRS and concentration CRS R6G ethanol solution was dispersed to an area of SRS on a clean quartz substrate, and dried to form R6G solid thin film. Thus Eq. (1) can be rewritten as follows: EF =
ISERS SSERS VRS CRS · IRS SRS VSERS CSERS
(2)
In the experiments, 10 L 10−11 M R6G solution was dispersed to an area of about 20 mm2 on G/AgNP array substrate and 60 L 10−4 M R6G solution was dispersed to an area of about 40 mm2 on the quartz substrate. Fig. S2 shows the SERS spectra and the normal Raman spectrum of R6G from the above-mentioned substrates. The intensities for the C C C in-plane bend mode at 612 cm−1 from G/AgNP, AgNP array substrates and solid R6G film on quartz substrate are 150, 60, 93 counts, respectively. According to the above equations, the average EF is calculated to be 4.8 × 107 for the G/AgNP array substrates. Similarly, the EF for the AgNP array substrates is calculated to be 1.9 × 106 . Compared with the bare AgNP array substrates, the G/AuNP array substrates shows an enhancement of 25-fold in the EF values. By considering the fact that the
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Fig. 3. (a) SEM top- and cross-section (inset) images of G/AuNP arrays. Inset image scale bar: 100 nm (b) Raman spectrum from the G/AuNP arrays.
additional CM enhancement from monolayer graphene was only 2–17 times [32], the improved sensitivity of the SERS substrate can be also attributed to the coupling of graphene and plasmonic AuNP arrays, graphene-induced fluorescence quenching and molecular absorption effect. Since graphene physically interact with Au surface, the narrow gap between graphene and Au surface will play important rule in producing SERS effect [49]. It has been reported that the maximum electrical field intensity around AuNP coated with monolayer graphene is approximately 1.3-fold higher than that around the bare AuNPs [34]. By comparing the electromagnetic field distribution of the AuNPs, the electromagnetic field of the graphene–AuNP hybrids is more confined in a narrower region about 3–5 nm around the interface between graphene and AuNP hybrids, due to the coupling of graphene and plasmonic AuNPs [34]. The delocalized -bond of graphene can serve as driving force for collecting target molecules by - interactions and making them tightly absorbed at the hot spots region near the interface [50]. The enhanced electromagnetic hot spots at the narrow interface region between graphene and Au nanoparticles make G/AuNP arrays higheffective surface-enhanced Raman scattering. By the multi-roles of graphene, the quality of SERS signals including sensitivity and signal-to-noise ratio were significantly improved. 3.4. Reproducibility and stability of the G/AuNP arrays Besides the high enhancement activity, the reproducibility of SERS spectra is another important issue for the use of SERS as a routine analytical tool. Raman intensity mappings of the R6G molecules at 10−7 M are measured over 60 m × 60 m to
Fig. 4. (a) Raman spectra of R6G molecules on AuNP array substrates with different concentrations from 10−7 to 10−10 . (b) Raman spectra of R6G molecules from G/AuNP array substrates with different concentrations from 10−7 to 10−11 .
evaluate the SERS signal reproducibility on the AgNP array substrates. The Raman data were obtained at every 1.0 m. Fig. 5a shows the Raman mapping of vibration modes at 612 cm−1 of R6G molecules dispensed on AuNP array substrates. The brighter colors represent higher intensities of the SERS signal and the dark colors represent lower intensities of the SERS signal. From the color scale in Fig. 5a, the Raman intensity shows a relatively large range from 195 to 256 counts. The maximum intensity deviation is evaluated to be about 13.5% based on the following formula: D=
I I − I¯ × 100% = × 100% I¯ I¯
(3)
where D represents the maximum intensity deviation, I is the maximum peak intensity and I¯ is the average peak intensity. In contrast, Raman mapping collected from the G/AuNP array substrates shows a more uniform color distribution as shown in Fig. 5b and the peak intensity lie between 1883 and 2140 counts. Based on formula (3), the intensity deviation is calculated to be about 6.4%. The narrow intensity distribution demonstrates that the SERS signals from G/AuNP array substrates have a high reproducibility over a continuous large area of 60 m × 60 m. Raman mappings of R6G with concentration reduced at 10−10 M and 10−11 M were also
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Fig. 5. SERS mapping of vibration modes at 612 cm−1 of R6G molecules at 10−7 M dispensed on the AuNP array substrates (a) and on (b) the G/AuNP array substrates over 50 m × 50 m area. (c) Average SERS spectrum of the R6G molecules from 20 positions on a same G/AuNP array substrate (red line). (d) Average SERS spectrum of the R6G molecular from 20 G/AuNP array substrates in different batches (blue line). The color shaded areas represent the deviations of the SERS spectra with respect to the average. (e) Intensity distribution of the 612 cm−1 peak in the 20 spectra from a same G/AuNP array substrate. (f) Intensity distribution of the 612 cm−1 peak in the 20 spectra from 20 G/AuNP array substrates in different batches. The color zones represent ±5% intensity variation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
measured on the G/AuNP array substrates (Fig. S3, in the Supporting information). Because of sharp decrease in molecular density, the pixel points in the mappings are not continuous. The individual pixel can be attributed to the spatial coincidence of the analyte molecules with the hot spots of the G/AuNP array substrates. The uniform distributed pixel points on the mappings demonstrate the homogenous distribution of both the analyte molecules and the hot spots on the G/AuNP arrays. The uniform distribution of absorbed molecules and hot spots can be attributed to the well-ordered structure of G/AuNP arrays (Fig. 3a) and good affinity of graphene for target molecules [50], which provides good explanation for the
high reproducibility of SERS signals of R6G at the higher concentration of 10−7 M. For practical application of SERS technology, what we concerned more is to obtain high reproducible SERS signal in millimeter-scale range or even from the SERS substrates in different batches [51]. The reproducibility of the SERS signals collected from random locations spacing more than 3 mm apart and from different substrates in various batches was evaluated. Fig. 5c shows the SERS spectra of R6G molecules at 10−7 M from 20 positions in a same G/AuNP substrate. These spectrum signals overlap to form a shaded area as filled with green. The red curve in the green shaded area is the average SERS spectrum of the 20 spectra of R6G molecules. The
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Table 1 Reproducibility analysis of SERS signals collected from different positions on a same substrate and from different substrates in various batches. Peak position (cm−1 )
612 773 1185 1362 1506 G 1647
From different positions on the same substrate
From different substrates in various batches
I¯ (counts)
I (counts)
D (%)
I¯ (counts)
I (counts)
D (%)
1779.50 1172.32 1034.40 3310.08 1320.24 2827.36 1861.92
118.34 80.79 52.34 162.94 90.30 133.95 113.78
6.65 6.89 5.06 4.92 6.84 4.74 6.11
1786.84 1153.36 1003.04 3408.12 1329.20 2896.24 1910.16
145.30 96.52 56.89 204.81 101.56 147.40 137.92
8.13 8.37 5.67 6.01 7.64 5.09 7.22
narrow shaded area (green color) indicates that the fluctuations of SERS signals for various vibration modes are all very small. The maximum intensity deviation of the SERS spectra with respect to the average intensity is given by formula (3). The maximum deviations of the typical peaks of R6G and G peak of graphene were listed in detail in Table 1. The peak intensities of spectra shows a minor fluctuation in the range of 4.74% to 6.89% depending on the different vibration modes. Fig. 5d shows the SERS signals of R6G molecules at 10−7 M on 20 different G/AuNP array substrates in various batches. Encouragingly, the fluctuation area (light blue) is also very narrow. As shown in Table 1, the small fluctuations of the SERS signals are limited to 9% for the entire vibration modes. To obtain a statistically meaningful result, the spot-to-spot intensity variation of the characteristic 612 cm−1 peak is quantified and presented in Fig. 5e. The red line indicates the average intensity of 20 spectra and the green zone represent ±5% intensity variation. Of the 20 data points, 18 lie within a 5% variation range and the other 2 are in the range of 5% to 7%. Fig. 5f shows the intensity variation of the 612 cm−1 peak of the spectra from 20 different G/AuNP array substrates in various batches. As shown by light blue zone, 17 lie within a 5% variation range and the other 3 are less than 9%. According to the scientific standards on the reproducibility of SERS substrates reported by Natan [51], the reproducibility of SERS signal from G/AuNP arrays has been much better than the requirements for quantitative measurements that the deviation of SERS signal from spot-to-spot or substrate-to-substrate should be less than 20%. Compared to the large fluctuations of SERS signal from AuNP array substrates, the high reproducibility of the SERS signals from G/AuNP array substrates can be attributed to at least three aspects: first, the well-transferred graphene layer effectively and homogenously captures the interested molecules by delocalized -bond of graphene, reducing the signal blinking induced by the drift of analyte molecules on SERS-active surfaces under excitation laser; second, the graphene layer reduces the additional signal by isolating the metal from the analyte molecules and enables the SERS analysis with more well-defined molecular interactions; third, the graphene layer can also effectively quench the background fluorescence, further improving the stability of the spectra. In fact, the absorption and isolation functions provided by graphene can also reduce photo-induced damage for SERS detection, which further improves the stability of Raman signals. The time-dependent experiments were carried out to compare the signal stability from AuNP arrays with that from G/AuNP arrays. As shown in Fig. 6, the SERS intensity of R6G on the bare AuNP arrays decayed quickly during a 450 s-long measurement, while the signals of R6G on G/AuNP arrays remained considerably more stable. Due to chemical stability of graphene, the stable graphene may serve as a passivation layer to suppress catalytic effect from Au nanoparticles, preventing R6G molecules from photo-induced carbonization. For practical applications, long-term stability is another important requirement for the SERS-active substrates. Therefore, the stability of the G/AgNP array substrates was also checked by comparing with ones stored in air for a long period of time. After
4 months of storage, the SERS intensity of R6G on the G/AuNP array substrates keeps almost the original intensity (Figs. S4 and S5, in the Supporting information), displaying a prominent advantage in terms of the stability. Long-term stability of G/AuNP array substrates is benefit from the very stable chemical properties of both gold and graphene. The high reproducibility and stability of SERS signal from R6G detection indicates that the G/AuNP array substrates have great potential for practical applications. 3.5. Practical applicability of the G/AuNP arrays To investigate the feasibility of the G/AuNP array substrates in practical application, the adenosine in pure water with concentration of 250 nM and in diluted human serum with different concentrations from 2 to 250 nM (one percent of serum in water) were tested on G/AuNP array substrates (Fig. 7a). Many peaks at about 725, 847, 1307 and 1337 cm−1 are observed. These peaks are all belong to adenosine according to the reported literatures [52–54]. Compared to the spectrum of adenosine in pure water (top green curve in Fig. 7a), only a few extremely weak and broad peaks in the range of 750 to 820 cm−1 and 1100 to 1250 cm−1 are added into the spectra of the samples in the spectrum. These
Fig. 6. Stability of the enhanced Raman signal of R6G molecules on (a) AuNP array substrates, and on (b) G/AuNP array substrates.
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kind of small molecule and it can be more readily absorbed on the narrow gaps of Au nanoparticles (hot spots) and thus more sensitive Raman signals are obtained. On the other hand, because of the heterocyclic aromatic structure of adenosine, the – interactions of graphene–adenosine can serve as a strong driving force to bring adenosine in close proximity to hot spot of G/AuNP array substrates [44], which can be another important factor for the high selectivity of adenosine. 4. Conclusions We have presented a kind of SERS-active substrate based on the hybrid structure of graphene and AuNP arrays. By the virtue of good plasmonic properties of AuNP arrays and unique physical/chemical properties of graphene, the G/AuNP array substrates show high performance in terms of sensitivity, signal-to-noise ratio and reproducibility. Using R6G molecules as a probe, the G/AuNP array substrates display excellent SERS activity with a minimum detected concentration of 10−11 M. The maximum deviations of SERS intensities from 20 positions of one SERS substrate are less than 7% and from 20 different substrates in various batches are less than 9%. On the basis of the G/AuNP arrays, a good linear relationship between SERS peak intensity and adenosine concentration is obtained in a range from 2 to 250 nM. This work may offer a novel and practical method to facilitate biosensing applications Acknowledgments The authors are grateful for financial support from the Shandong Province Natural Science Foundation (ZR2014FQ032 and BS2014CL039) and National Natural Science Foundation of China (61205174, 61401258, and 11404193). Fig. 7. (a) Raman spectra of adenosine in pure water (top green curve) with concentration of 250 nM and in diluted human serum with different concentrations from 2 to 250 nM tested on G/AuNP array substrates. (b) Raman intensity of adenosine in diluted serum at 1337 cm−1 as a function of the adenosine concentration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
weak and broad peaks can be regarded as signals from serum in comparison with the spectrum from blank serum (bottom black curve in Fig. 7a). This fact indicates that the main peaks in the spectra collected from compounds of adenosine in serum are actually from adsorbed adenosine, and are not derived from the serum. The lowest detected concentration of adenosine in serum is 2 nM as shown in red curve in Fig. 7a. To show the capability of the quantitative detection of adenosine in serum and its reproducibility, the linear fit calibration curve (R2 = 0.995) with error bars is illustrated in Fig. 7b. The intensities of the SERS spectra of adenosine are proportional to the logarithm of the concentrations of the adenosine in diluted serum. The good linear response of SERS is obtained from 2 to 250 nM. According to the error bars, the maximum deviation was calculated to be about 5.3% with respect to the concentration at 70 nM. The small deviation further conform the good reproducibility of the G/AuNP array substrates. The ignorable protein background and the good line correlation indicate a great potential application of the G/AuNP array substrates to detect other analytes in real biological systems. The high selectivity for adenosine with respect to the complicated protein matrices in human serum is compelling. Because of the finite number of SERS-active sites, the competition for surface active sites is a very real concern. Compared to the complicated protein matrices in human serum, adenosine is a
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Biographies Shicai Xu is a lecturer in the Dezhou University. He received his Ph.D. at College of Physics and Electronics, Shandong Normal University in 2014. His research interest is preparation and application of graphene and its derivatives. Shouzhen Jiang obtained his Ph.D. degree from Shandong University in 2007. He then worked as a university lecturer in Shandong Normal University. His research interests are mainly centered on studying optical properties of Dirac materials. Jihua Wang is a professor in Dezhou University. He obtained his Ph.D. degree from Shandong University in 2003. His research interests are mainly centered on biological functional macromolecules and gene chip. Jie Wei is an attending doctor in Dezhou People’s Hospital. She obtained her Ph.D. degree from Central South University in 2008. Her research interests are mainly centered on surface-enhanced Raman scattering. Weiwei Yue is a lecturer in the Shandong Normal University. He received his Ph.D. at the State Key Laboratory of Transducer Technology, Institute of Electronics CAS in 2008. His research interests have been on graphene biosensors, optic and electrical detection and instrumentation for biosensors. Yong Ma is a lecturer in the Shandong Normal University. He received his Ph.D. at College of Physics and Electronics, Shandong Normal University in 2014. His research interests have been on theoretical study of X-ray spectroscopy and Raman spectroscopy for functional organic materials and graphene.
Please cite this article in press as: S. Xu, et al., Graphene isolated Au nanoparticle arrays with high reproducibility for high-performance surface-enhanced Raman scattering, Sens. Actuators B: Chem. (2015), http://dx.doi.org/10.1016/j.snb.2015.08.009
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Graphene isolated Au nanoparticle arrays with high reproducibility for high-performance surface-enhanced Raman scattering Shicai Xu a,∗ , Shouzhen Jiang b , Jihua Wang a , Jie Wei c , Weiwei Yue b , Yong Ma b a College of Physics and Electronic Information, Shandong Provincial Key Laboratory of Functional Macromolecular Biophysics, Dezhou University, Dezhou 253023, China b College of Physics and Electronics, Shandong Normal University, Jinan 250014, China c Department of Neurology, Dezhou People’s Hospital, Dezhou 253014, China
a r t i c l e
i n f o
Article history: Received 17 June 2015 Received in revised form 2 August 2015 Accepted 5 August 2015 Available online xxx Keywords: Graphene Au nanoparticle arrays Nanostructure SERS Adenosine
a b s t r a c t An efficient surface enhanced Raman scattering (SERS) substrate has been developed based on highly ordered arrays of graphene–isolated Au nanoparticle (G/AuNP). By combining the electromagnetic enhancement activity of AuNP arrays and unique physical/chemical properties of graphene, the G/AuNP arrays shows high performance in terms of sensitivity, signal-to-noise ratio and reproducibility. The average enhancement factor obtained from the G/AuNP arrays for rhodamine 6G probe molecules is over 107 . The maximum deviations of SERS intensities from 20 positions of a same SERS substrate are in the range of 4.74% to 6.89% and from 20 different substrates in various batches are in the range of 5.67% to 8.37%, depending on different vibration modes. As a practical application of this SERS system, we detect the adenosine concentration in human serum. The detection results show a good linear correlation between SERS intensity and adenosine concentration within the range of 2 to 250 nM. This work may open up new opportunities in developing the applications of SERS in biomedical diagnostics, biological sensing and other biotechnology. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Surface-enhanced Raman scattering spectroscopy is a powerful technique for ultrasensitive and selective detection in biology and chemistry [1–3]. Although Raman spectroscopy is limited by low sensitivity, SERS can provide signal intensity of the molecules enhanced by orders of magnitude on the proper substrates. Though the detailed physical mechanism of SERS is still under debate, it has been widely accepted that SERS enhancement results from a combination of an electromagnetic mechanism (EM) and a chemical mechanism (CM) [4]. The EM originates from the dramatic increase in the local electromagnetic field due to a light-excited surface plasmon resonance, which can boost the pristine Raman signal by 108 times or more [5,6]. Yet for CM, it is caused by charge transfer between the target molecules and the substrate, usually has a minor enhancement factor of 10–102 [7]. Both experimental and theoretical results indicate that the EM enhancement of SERS is from metal nanoparticles (NPs) and the enhancement factor depends on the gap between adjacent NPs [8–10]. A great deal of research
∗ Corresponding author. Tel.: +86 5348982081. E-mail address: [email protected] (S. Xu).
effort in SERS community has been focused on the fabrication of controlled and reproducible metallic nanostructures incorporating as much as possible the hot spots, including the colloidal clusters of noble metallic NPs [11–13], surface-roughened noble metals [14], and well-defined top-down fabricated metal architectures [15,16], such as Ag nanoclusters on ZnO nanodome arrays [17] well-ordered Au-nanorod arrays [18], Au nanorods coupled with Ag nanoparticles [19]. During the past four decades, great efforts and ingenuity have gone toward creating a more desirable SERS substrate, resulting in extraordinary sensitivity ultrasensitive [20,21], or even at the single-molecule level [22–24]. Yet SERS has not entered the widespread real world application stage. The main problem is that the general SERS substrates based on Ag and Au, either in the form of dispersed nanoparticles or ordered nanoparticles, nanowires, and nanorod arrays are difficult to release high reproducible SERS signals for facing many challenges: first, it is required to have a large-area SERS substrate with regularly arranged nanostructures containing well-controlled narrow gaps that can generate the homogeneous hot spots; second, it is also required to capture the interested molecules effectively and homogenously on the surface of the SERS substrate; third, the large fluctuations of SERS signal need to be minimized. The sources of fluctuations include dynamic interaction of the target molecules with the local environment of plasmonic metal [25,26], the change of the adsorption
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sites caused by the restructuring of the substrate surface [27], the drift of analyte molecules on substrate surface caused by excitation laser [14]. These challenges cause low SERS signal even in the most sophisticated measurements. Because of the poor reproducibility, the practicality of the SERS has always been questioned by the non-SERS community. While people have strived to deal with the first challenge to create reproducible structures by fabricating well-defined SERS active substrates [8,15], the latter problems may be equally important but ignored to some extent. Recently, a thin and pinhole free layer of SiO2 or Al2 O3 as an inert shell was employed in the SERS substrates to isolate metal nanostructures from their surroundings [28]. This strategy shows one overwhelmingly attractive feature that the SERS signal derives only from the target analytes and spurious signals induced by the metal is locked by inert shell. Fabrication of SERS substrates with an ultrathin passivated surface at a lowest loss of electromagnetic enhancement activity is the key to shell-isolated SERS. The main challenge is to get a pinhole-free coating layer with a very small thickness. Graphene, a 2D atomic crystal with densely packed carbon atoms in a honeycomb crystal lattice [29], is well-known for its unique electrical performance and the amazing applications in nanoscale electronics [30]. As well as the wide use in electronics field, the atomic thickness and seamless structure of graphene also makes it a natural candidate material for shell-isolated SERS [31]. The ultrathin graphene layer can provide effective transfer of the electromagnetic field from the metal core through the shell to the probe molecule, at the same time the stable and pinhole-free shell can protect the inner metal core and prevent the direct metal–molecule contact. Besides the isolation function like SiO2 or Al2 O3 shell, graphene layer as a new kind of shell also has many other functions. Typical functions include surface adsorption [32], additional chemical enhancement and fluorescence quenching [33]. To take advantage of these functions of graphene, many efforts have been done to fabricate graphene-isolated metallic nanostructure for SERS. Xu et al. prepared a graphene–veiled gold substrate with a passivated surface for SERS [14]. Xie’s group fabricated a hybrid SERS-active platform consisting of a single layer graphene covering a quasiperiodic Au nanopyramid that enabled single molecule detection for rhodamine 6G (R6G) [31]. Zhu’s group has shown that graphene–gold nanoparticle hybrid films is a promising material for shell isolated SERS [34]. Li et al. fabricated monolayer graphenecovered Ag nanoparticles and demonstrated that the graphene layer can obviously suppress the oxidation of Ag nanoparticles [35]. These works provided facile methods to use graphene to wrap metal nanoparticles and more stable SERS signals were obtained. However, in these studies, either for the colloidal clusters of noble metallic NPs [34,35] or surface-roughened noble metals [14], the hot spots are randomly distributed on the SERS-active substrates. Although the graphene as a kind of additive has been employed in SERS substrates, high reproducible SERS signals are still difficult to achieve due to the spatial inhomogeneity of these plasmonic substrates. Thus it is necessary to design a kind of SERS substrate to obtain high reproducible SERS signals by combining the high ordered structure of plasmonic metal nano arrays with the multifunctions of graphene. Herein, we demonstrate that clean and high reproducible SERS signals were obtained by combining large-area AuNP arrays with monolayer graphene. The G/AuNP arrays boost a high density of hot spots with average SERS enhancement factor over 107 for R6G detection. The added graphene layer enables SERS analysis with more well-defined molecular interactions and thus improves the reproducibility of SERS signals. The maximum fluctuations from 20 positions of a same SERS substrate are less than 7% and from 20 different substrates in various batches are less than 9%. The high reproducibility of the enhanced Raman signals can be attributed to the well-ordered morphology of AuNP arrays and
multi-functions of graphene layer including metal–molecule isolation, surface adsorption, and fluorescence quenching. On the basis of the AuNP array substrates, the adenosine SERS signals with high selectivity and reproducibility were detected in human serum. Adenosine is the metabolite of adenine nucleotides, which has been regarded as one of the major neuromodulators. As the core molecule of ATP and of nucleic acids, adenosine forms a unique link among cell energy, gene regulation, and neuronal excitability [36]. The successful detection of adenosine in human serum indicated that the G/AuNP array substrates have great potential for detecting other analytes in real biological systems. 2. Experimental 2.1. Materials and instruments R6G and adenosine were purchased from Aladdin Industrial Corp. Ltd (Shanghai, China) as test samples. Human serum was provided by Department of Internal Medicine of Dezhou People’s Hospital for the feasibility test of the G/AuNP array substrates. The morphologies of AuNP arrays were characterized by scanning electron microscopy (SEM, ZEISS, SUPRATM-55). The transmission electron microscopy (TEM) images were obtained by a transmission electron microscope system (JEOL JEM-2100) with X-ray energy dispersion analysis equipment (EDS, JEOL Company). For TEM analysis, the quartz-supported AuNP arrays was immersed in 35% HF solution for 2 h to free individual particles from the substrates. The SERS experiments and the graphene characterization were carried out in a Raman spectrometer (Horiba HR-800) with laser excitation at 532 nm. 2.2. Preparation of AuNP arrays The AuNP arrays were fabricated by electron beam evaporation using anodic alumimum oxide (AAO) as template. The throughpore AAO templates were prepared via a two-step anodization of pure aluminum foils (99.999%). The first anodization was performed under constant potentials of 40 V in 0.3 M oxalic acid at 20 ◦ C for 7 h. Secondary anodization was carried out under constant potentials of 40 V in 0.4 M phosphoric acid at 20 ◦ C for 10 min. After the barrier layer was removed, the pores were widened via wet chemical etching in 5 wt% phosphoric acid at 30 ◦ C for 40 min. By using through-pore AAO as template, a gold layer of about 100 nm was evaporated onto the quartz substrates. Finally, the AAO template was completely removed by immersing in 5% phosphoric acid to expose the AuNP arrays on the quartz substrates. 2.3. Preparation of monolayer graphene The large-area graphene was synthesized on a piece of copper foils (25 m thick) in a quartz tube at 1050 ◦ C by chemical vapor deposition (CVD) as reported in detail in our previously study [37]. First, the copper foils were annealed for 10 min with flowing hydrogen of 15 sccm at 90 Pa to increase the copper grain size [38]. Then, the gas mixture of methane (10 sccm) and hydrogen (65 sccm) was introduced into quartz tube at 200 Pa for 20 min to grow graphene. Finally, the samples were rapidly cooled to room temperature with flowing hydrogen at rate of 65 sccm. 2.4. Preparation of G/AuNP arrays The preparation processes of G/AuNP arrays are summarized in Fig. 1. By using CVD method, large-area graphene was grown on a piece of Cu foil (Fig. 1a and b). Polymethylmethacrylate (PMMA) was spincoated on one side of the foil and the Cu foil was subsequently etched away using 0.1 M aqueous FeCl3 solution for 20 min
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Fig. 1. Preparation processes of G/AuNP array substrates.
(Fig. 1c and d). Then the graphene/PMMA film was washed with HCl/H2 O (1:10) and deionized water for three times and then transferred onto the quartz-supported AuNP arrays (Fig. 1e). To avoid the breakage of the graphene, we employed a small amount of liquid PMMA to introduce a second PMMA coating on the precoated PMMA/graphene/AuNP arrays. The redissolution of the precoated PMMA tends to mechanically relax the underlying graphene [39], leading to a better contact with the surface morphology of the underlying AuNP arrays (Fig. 1f and g). Then the PMMA layer completely removed by being dissolved in dichloromethane (DCM) at 50 ◦ C within several minutes (Fig. 1h). Finally, the substrate was rinsed with isopropyl alcohol and the graphene isolated AuNP array substrates were formed (Fig. 1i).
nanodots after removal of the AAO template. The hexagonally arranged Au particles are almost identical in size and topology. The average diameter of the particles is ∼90 nm, and the distance between two neighboring particles is ∼60 nm. The period of the nanoparticle array is ∼150 nm, which is consistent with the period of the AAO template. Fig. 2c shows the TEM image of Au particles that have been completely liberated from the quartz substrates. The image shows that the Au particles are spherical and the average diameter of the particles is ∼90 nm, in good agreement with the result of the SEM images. Fig. 2d shows EDS spectrum from the Au particles. All the peaks are associated to Au element, demonstrating a high purity of Au particles. 3.2. Characterization of G/AuNP array substrates
2.5. SERS experiments SERS experiments were carried out using a Raman system (Horiba HR-800) with laser excitation at 532 nm. The excitation laser spot was about 1 m, and the effective power of the laser source was kept at 0.05 mW for R6G molecules, and 0.25 mW for adenosine molecules. The system was connected to a microscope, and the laser light was coupled through an objective lens of 20×, which was used for exciting the samples as well as collecting the Raman signals. Prior to each Raman experiment, calibration of the instrument was done with the Raman signal from a silicon standard centered at 520 cm−1 . SERS substrates were incubated for 3 h in different analyte solutions and then were placed into a vacuum drying oven to evaporate any moisture. The substrates were taken out and fixed onto the glass slide for SERS measurements. The SERS measurements were performed from at least 8 random locations that were more than 3 mm apart with an accumulation time of 10 s. If there is no special instruction, the mentioned Raman spectra are expressed in terms of average spectra. 3. Results and discussion 3.1. Characterization of AuNP arrays Fig. 2a shows the SEM image of a typical AAO membrane. The typical area of the nanohole arrays is defect-free and these nanoholes with uniform size are hexagonally arranged in high regularity. The mean diameter and the interval of the holes are ∼100 nm and ∼50 nm, respectively. The AuNP arrays were fabricated through a standard electron beam evaporation technique using the AAO template. Fig. 2b shows a representative SEM image of Au
Fig. 3a shows the SEM top- and cross-section (inset) images of the hybrid structure of G/AuNP array substrates. Graphene deforms itself to conform to the AuNP geometry and only a few of breakages is observed as marked by the white arrows. The cross-section image shows a clear lateral-view of G/AuNP arrays, consistent with the schematic structure of G/AuNP arrays (Fig. 1). As both SEM top- and cross-section images cannot give direct evidence of graphene, a conventional Raman measurement was performed on these hybrids to demonstrate the existence of graphene. As shown in Fig. 3b, the D, G and 2D peaks of graphene are clearly observed at ∼1360, ∼1580 and ∼2695 cm−1 , respectively. These peaks can be regarded as the fingerprint of graphene [40,41]. The Raman spectrum shows typical features of monolayer graphene: the intensity ratio of I(2D) /I(G) ≥ 2 and a single Lorentzian 2D peak with a full width at half maximum (FWHM) of ∼35 cm−1 [42]. Here, the defectrelated D peak is very weak, indicative of high quality of graphene. These results demonstrate that the graphene film with a monolayer structure is actually transferred on AuNP arrays. To evaluate the uniformity of graphene, Raman mappings of graphene on AuNP arrays were implemented over 60 m × 60 m area as shown in Fig. S1 in the Supporting information. From the color scale in Fig. S1a, it can be seen that the intensity ratios of 2D-peak to G-peak (I2D /IG ) show a very small fluctuation from 2.6 to 2.8, indicating that the monolayer graphene have a very uniform structure in a large area. Disorder-induced D-peaks were also detected with the intensity ratio of the D-peak to G-peak (ID /IG ). The Raman ID /IG ratio was extensively used to estimate the quality of graphene and the degree of disorder. As shown in Fig. S1b, the intensity ratios of ID /IG are less than 0.07, indicating that the employed graphene has little defects even in a large area.
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Fig. 2. (a) SEM image of through-pore AAO membrane. (b) SEM image of AuNP arrays. (c) TEM image of Au particles liberated from quartz substrate. (d) EDS spectrum of the Au particles.
3.3. SERS activity of G/AuNP arrays To verify the SERS activity of G/AuNP array substrates, we compared the performance of G/AuNP array substrates with that of AuNP array substrates. By maintaining the same measurement conditions, the R6G aqueous solutions with varied concentrations were used as the probe molecule. Fig. 4a shows the SERS spectra of R6G molecules from AuNP array substrates with various concentrations of 10−7 , 10−8 , 10−9 , 10−10 M, respectively. The Raman peaks at 612, 773, 1185, 1311, 1362, 1506, 1570 and 1647 cm−1 are in good agreement with previous reports for R6G [43,44]. Beside of characteristic peaks of R6G, some additional weak peaks are also observed as marked by red arrows. These additional peaks are difficult to be assigned precisely, which can be attributed to the various possible interactions between AuNPs and the R6G molecules, including charge transfer between the metal and molecules, photo-induced damage and metal-catalyzed side reactions, etc. [27]. The quality of SERS signals in terms of sensitivity and signal-to-noise ratio was improved on the G/AuNP arrays. As shown in Fig. 4b, the additional weak peaks and fluorescence background in the SERS spectra are obviously reduced and the characteristic signals of R6G are still visible even at 10−11 M. Because of the additional graphene layer, the R6G molecules are isolated from metal AuNP arrays and the spurious SERS signals from various possible metal–molecule interactions are thus reduced. Though a few of graphene breakages is observed on the G/AuNP arrays, the additional graphene layer still shows great isolation effect. That’s probably because most of the hot spot is located in between two consecutive AuNPs, whereas few graphene breakages exist in this region as shown in Fig. 3a. The reduction of fluorescence background can be explained by the fluorescence quenching effect of graphene. This effect is basically due to a resonance energy transfer process from dye molecules to graphene as reported from Swathi et al. [45]. The G peaks of
graphene are also clearly observed, which are nearly independent of the concentrations of R6G. The SERS enhancement factors (EF) for R6G on the G/AuNP arrays were calculated according to the standard equation [46–48]: EF =
ISERS /NSERS IRS /NRS
(1)
where ISERS and IRS , respectively, represent the peak intensities of the SERS spectra and the normal Raman spectra, NSERS and NRS are, respectively, the numbers of molecules on the substrates within the laser spot. In the experiments, a certain volume VSERS and concentration CSERS R6G aqueous solution was dispersed to an area of SSERS on the SERS-active substrates. For normal Raman experiment, a certain volume VRS and concentration CRS R6G ethanol solution was dispersed to an area of SRS on a clean quartz substrate, and dried to form R6G solid thin film. Thus Eq. (1) can be rewritten as follows: EF =
ISERS SSERS VRS CRS · IRS SRS VSERS CSERS
(2)
In the experiments, 10 L 10−11 M R6G solution was dispersed to an area of about 20 mm2 on G/AgNP array substrate and 60 L 10−4 M R6G solution was dispersed to an area of about 40 mm2 on the quartz substrate. Fig. S2 shows the SERS spectra and the normal Raman spectrum of R6G from the above-mentioned substrates. The intensities for the C C C in-plane bend mode at 612 cm−1 from G/AgNP, AgNP array substrates and solid R6G film on quartz substrate are 150, 60, 93 counts, respectively. According to the above equations, the average EF is calculated to be 4.8 × 107 for the G/AgNP array substrates. Similarly, the EF for the AgNP array substrates is calculated to be 1.9 × 106 . Compared with the bare AgNP array substrates, the G/AuNP array substrates shows an enhancement of 25-fold in the EF values. By considering the fact that the
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Fig. 3. (a) SEM top- and cross-section (inset) images of G/AuNP arrays. Inset image scale bar: 100 nm (b) Raman spectrum from the G/AuNP arrays.
additional CM enhancement from monolayer graphene was only 2–17 times [32], the improved sensitivity of the SERS substrate can be also attributed to the coupling of graphene and plasmonic AuNP arrays, graphene-induced fluorescence quenching and molecular absorption effect. Since graphene physically interact with Au surface, the narrow gap between graphene and Au surface will play important rule in producing SERS effect [49]. It has been reported that the maximum electrical field intensity around AuNP coated with monolayer graphene is approximately 1.3-fold higher than that around the bare AuNPs [34]. By comparing the electromagnetic field distribution of the AuNPs, the electromagnetic field of the graphene–AuNP hybrids is more confined in a narrower region about 3–5 nm around the interface between graphene and AuNP hybrids, due to the coupling of graphene and plasmonic AuNPs [34]. The delocalized -bond of graphene can serve as driving force for collecting target molecules by - interactions and making them tightly absorbed at the hot spots region near the interface [50]. The enhanced electromagnetic hot spots at the narrow interface region between graphene and Au nanoparticles make G/AuNP arrays higheffective surface-enhanced Raman scattering. By the multi-roles of graphene, the quality of SERS signals including sensitivity and signal-to-noise ratio were significantly improved. 3.4. Reproducibility and stability of the G/AuNP arrays Besides the high enhancement activity, the reproducibility of SERS spectra is another important issue for the use of SERS as a routine analytical tool. Raman intensity mappings of the R6G molecules at 10−7 M are measured over 60 m × 60 m to
Fig. 4. (a) Raman spectra of R6G molecules on AuNP array substrates with different concentrations from 10−7 to 10−10 . (b) Raman spectra of R6G molecules from G/AuNP array substrates with different concentrations from 10−7 to 10−11 .
evaluate the SERS signal reproducibility on the AgNP array substrates. The Raman data were obtained at every 1.0 m. Fig. 5a shows the Raman mapping of vibration modes at 612 cm−1 of R6G molecules dispensed on AuNP array substrates. The brighter colors represent higher intensities of the SERS signal and the dark colors represent lower intensities of the SERS signal. From the color scale in Fig. 5a, the Raman intensity shows a relatively large range from 195 to 256 counts. The maximum intensity deviation is evaluated to be about 13.5% based on the following formula: D=
I I − I¯ × 100% = × 100% I¯ I¯
(3)
where D represents the maximum intensity deviation, I is the maximum peak intensity and I¯ is the average peak intensity. In contrast, Raman mapping collected from the G/AuNP array substrates shows a more uniform color distribution as shown in Fig. 5b and the peak intensity lie between 1883 and 2140 counts. Based on formula (3), the intensity deviation is calculated to be about 6.4%. The narrow intensity distribution demonstrates that the SERS signals from G/AuNP array substrates have a high reproducibility over a continuous large area of 60 m × 60 m. Raman mappings of R6G with concentration reduced at 10−10 M and 10−11 M were also
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Fig. 5. SERS mapping of vibration modes at 612 cm−1 of R6G molecules at 10−7 M dispensed on the AuNP array substrates (a) and on (b) the G/AuNP array substrates over 50 m × 50 m area. (c) Average SERS spectrum of the R6G molecules from 20 positions on a same G/AuNP array substrate (red line). (d) Average SERS spectrum of the R6G molecular from 20 G/AuNP array substrates in different batches (blue line). The color shaded areas represent the deviations of the SERS spectra with respect to the average. (e) Intensity distribution of the 612 cm−1 peak in the 20 spectra from a same G/AuNP array substrate. (f) Intensity distribution of the 612 cm−1 peak in the 20 spectra from 20 G/AuNP array substrates in different batches. The color zones represent ±5% intensity variation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
measured on the G/AuNP array substrates (Fig. S3, in the Supporting information). Because of sharp decrease in molecular density, the pixel points in the mappings are not continuous. The individual pixel can be attributed to the spatial coincidence of the analyte molecules with the hot spots of the G/AuNP array substrates. The uniform distributed pixel points on the mappings demonstrate the homogenous distribution of both the analyte molecules and the hot spots on the G/AuNP arrays. The uniform distribution of absorbed molecules and hot spots can be attributed to the well-ordered structure of G/AuNP arrays (Fig. 3a) and good affinity of graphene for target molecules [50], which provides good explanation for the
high reproducibility of SERS signals of R6G at the higher concentration of 10−7 M. For practical application of SERS technology, what we concerned more is to obtain high reproducible SERS signal in millimeter-scale range or even from the SERS substrates in different batches [51]. The reproducibility of the SERS signals collected from random locations spacing more than 3 mm apart and from different substrates in various batches was evaluated. Fig. 5c shows the SERS spectra of R6G molecules at 10−7 M from 20 positions in a same G/AuNP substrate. These spectrum signals overlap to form a shaded area as filled with green. The red curve in the green shaded area is the average SERS spectrum of the 20 spectra of R6G molecules. The
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Table 1 Reproducibility analysis of SERS signals collected from different positions on a same substrate and from different substrates in various batches. Peak position (cm−1 )
612 773 1185 1362 1506 G 1647
From different positions on the same substrate
From different substrates in various batches
I¯ (counts)
I (counts)
D (%)
I¯ (counts)
I (counts)
D (%)
1779.50 1172.32 1034.40 3310.08 1320.24 2827.36 1861.92
118.34 80.79 52.34 162.94 90.30 133.95 113.78
6.65 6.89 5.06 4.92 6.84 4.74 6.11
1786.84 1153.36 1003.04 3408.12 1329.20 2896.24 1910.16
145.30 96.52 56.89 204.81 101.56 147.40 137.92
8.13 8.37 5.67 6.01 7.64 5.09 7.22
narrow shaded area (green color) indicates that the fluctuations of SERS signals for various vibration modes are all very small. The maximum intensity deviation of the SERS spectra with respect to the average intensity is given by formula (3). The maximum deviations of the typical peaks of R6G and G peak of graphene were listed in detail in Table 1. The peak intensities of spectra shows a minor fluctuation in the range of 4.74% to 6.89% depending on the different vibration modes. Fig. 5d shows the SERS signals of R6G molecules at 10−7 M on 20 different G/AuNP array substrates in various batches. Encouragingly, the fluctuation area (light blue) is also very narrow. As shown in Table 1, the small fluctuations of the SERS signals are limited to 9% for the entire vibration modes. To obtain a statistically meaningful result, the spot-to-spot intensity variation of the characteristic 612 cm−1 peak is quantified and presented in Fig. 5e. The red line indicates the average intensity of 20 spectra and the green zone represent ±5% intensity variation. Of the 20 data points, 18 lie within a 5% variation range and the other 2 are in the range of 5% to 7%. Fig. 5f shows the intensity variation of the 612 cm−1 peak of the spectra from 20 different G/AuNP array substrates in various batches. As shown by light blue zone, 17 lie within a 5% variation range and the other 3 are less than 9%. According to the scientific standards on the reproducibility of SERS substrates reported by Natan [51], the reproducibility of SERS signal from G/AuNP arrays has been much better than the requirements for quantitative measurements that the deviation of SERS signal from spot-to-spot or substrate-to-substrate should be less than 20%. Compared to the large fluctuations of SERS signal from AuNP array substrates, the high reproducibility of the SERS signals from G/AuNP array substrates can be attributed to at least three aspects: first, the well-transferred graphene layer effectively and homogenously captures the interested molecules by delocalized -bond of graphene, reducing the signal blinking induced by the drift of analyte molecules on SERS-active surfaces under excitation laser; second, the graphene layer reduces the additional signal by isolating the metal from the analyte molecules and enables the SERS analysis with more well-defined molecular interactions; third, the graphene layer can also effectively quench the background fluorescence, further improving the stability of the spectra. In fact, the absorption and isolation functions provided by graphene can also reduce photo-induced damage for SERS detection, which further improves the stability of Raman signals. The time-dependent experiments were carried out to compare the signal stability from AuNP arrays with that from G/AuNP arrays. As shown in Fig. 6, the SERS intensity of R6G on the bare AuNP arrays decayed quickly during a 450 s-long measurement, while the signals of R6G on G/AuNP arrays remained considerably more stable. Due to chemical stability of graphene, the stable graphene may serve as a passivation layer to suppress catalytic effect from Au nanoparticles, preventing R6G molecules from photo-induced carbonization. For practical applications, long-term stability is another important requirement for the SERS-active substrates. Therefore, the stability of the G/AgNP array substrates was also checked by comparing with ones stored in air for a long period of time. After
4 months of storage, the SERS intensity of R6G on the G/AuNP array substrates keeps almost the original intensity (Figs. S4 and S5, in the Supporting information), displaying a prominent advantage in terms of the stability. Long-term stability of G/AuNP array substrates is benefit from the very stable chemical properties of both gold and graphene. The high reproducibility and stability of SERS signal from R6G detection indicates that the G/AuNP array substrates have great potential for practical applications. 3.5. Practical applicability of the G/AuNP arrays To investigate the feasibility of the G/AuNP array substrates in practical application, the adenosine in pure water with concentration of 250 nM and in diluted human serum with different concentrations from 2 to 250 nM (one percent of serum in water) were tested on G/AuNP array substrates (Fig. 7a). Many peaks at about 725, 847, 1307 and 1337 cm−1 are observed. These peaks are all belong to adenosine according to the reported literatures [52–54]. Compared to the spectrum of adenosine in pure water (top green curve in Fig. 7a), only a few extremely weak and broad peaks in the range of 750 to 820 cm−1 and 1100 to 1250 cm−1 are added into the spectra of the samples in the spectrum. These
Fig. 6. Stability of the enhanced Raman signal of R6G molecules on (a) AuNP array substrates, and on (b) G/AuNP array substrates.
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kind of small molecule and it can be more readily absorbed on the narrow gaps of Au nanoparticles (hot spots) and thus more sensitive Raman signals are obtained. On the other hand, because of the heterocyclic aromatic structure of adenosine, the – interactions of graphene–adenosine can serve as a strong driving force to bring adenosine in close proximity to hot spot of G/AuNP array substrates [44], which can be another important factor for the high selectivity of adenosine. 4. Conclusions We have presented a kind of SERS-active substrate based on the hybrid structure of graphene and AuNP arrays. By the virtue of good plasmonic properties of AuNP arrays and unique physical/chemical properties of graphene, the G/AuNP array substrates show high performance in terms of sensitivity, signal-to-noise ratio and reproducibility. Using R6G molecules as a probe, the G/AuNP array substrates display excellent SERS activity with a minimum detected concentration of 10−11 M. The maximum deviations of SERS intensities from 20 positions of one SERS substrate are less than 7% and from 20 different substrates in various batches are less than 9%. On the basis of the G/AuNP arrays, a good linear relationship between SERS peak intensity and adenosine concentration is obtained in a range from 2 to 250 nM. This work may offer a novel and practical method to facilitate biosensing applications Acknowledgments The authors are grateful for financial support from the Shandong Province Natural Science Foundation (ZR2014FQ032 and BS2014CL039) and National Natural Science Foundation of China (61205174, 61401258, and 11404193). Fig. 7. (a) Raman spectra of adenosine in pure water (top green curve) with concentration of 250 nM and in diluted human serum with different concentrations from 2 to 250 nM tested on G/AuNP array substrates. (b) Raman intensity of adenosine in diluted serum at 1337 cm−1 as a function of the adenosine concentration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
weak and broad peaks can be regarded as signals from serum in comparison with the spectrum from blank serum (bottom black curve in Fig. 7a). This fact indicates that the main peaks in the spectra collected from compounds of adenosine in serum are actually from adsorbed adenosine, and are not derived from the serum. The lowest detected concentration of adenosine in serum is 2 nM as shown in red curve in Fig. 7a. To show the capability of the quantitative detection of adenosine in serum and its reproducibility, the linear fit calibration curve (R2 = 0.995) with error bars is illustrated in Fig. 7b. The intensities of the SERS spectra of adenosine are proportional to the logarithm of the concentrations of the adenosine in diluted serum. The good linear response of SERS is obtained from 2 to 250 nM. According to the error bars, the maximum deviation was calculated to be about 5.3% with respect to the concentration at 70 nM. The small deviation further conform the good reproducibility of the G/AuNP array substrates. The ignorable protein background and the good line correlation indicate a great potential application of the G/AuNP array substrates to detect other analytes in real biological systems. The high selectivity for adenosine with respect to the complicated protein matrices in human serum is compelling. Because of the finite number of SERS-active sites, the competition for surface active sites is a very real concern. Compared to the complicated protein matrices in human serum, adenosine is a
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Biographies Shicai Xu is a lecturer in the Dezhou University. He received his Ph.D. at College of Physics and Electronics, Shandong Normal University in 2014. His research interest is preparation and application of graphene and its derivatives. Shouzhen Jiang obtained his Ph.D. degree from Shandong University in 2007. He then worked as a university lecturer in Shandong Normal University. His research interests are mainly centered on studying optical properties of Dirac materials. Jihua Wang is a professor in Dezhou University. He obtained his Ph.D. degree from Shandong University in 2003. His research interests are mainly centered on biological functional macromolecules and gene chip. Jie Wei is an attending doctor in Dezhou People’s Hospital. She obtained her Ph.D. degree from Central South University in 2008. Her research interests are mainly centered on surface-enhanced Raman scattering. Weiwei Yue is a lecturer in the Shandong Normal University. He received his Ph.D. at the State Key Laboratory of Transducer Technology, Institute of Electronics CAS in 2008. His research interests have been on graphene biosensors, optic and electrical detection and instrumentation for biosensors. Yong Ma is a lecturer in the Shandong Normal University. He received his Ph.D. at College of Physics and Electronics, Shandong Normal University in 2014. His research interests have been on theoretical study of X-ray spectroscopy and Raman spectroscopy for functional organic materials and graphene.
Please cite this article in press as: S. Xu, et al., Graphene isolated Au nanoparticle arrays with high reproducibility for high-performance surface-enhanced Raman scattering, Sens. Actuators B: Chem. (2015), http://dx.doi.org/10.1016/j.snb.2015.08.009