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Abstract
Quantitative analysis is of importance for surface-enhanced Raman scattering (SERS). However, due to fluctuations in the enhancing performance of the substrates, it is difficult to obtain reliable results. In this paper, a reliable quantitative method is introduced to overcome this problem with graphene on the top of Ag nanoholes structure as SERS substrates by an internal standard method. To achieve the internal standard method, Ag nanoholes are firstly prepared by surface plasmon (SP) lithography technology. Then a monolayer graphene is transferred onto the surface of the Ag nanoholes structure. 2D Raman peak of graphene is used as an internal standard to normalize the intensity of analyte molecules. The random representative and averaged Raman intensity of different concentration of rhodamine 6G (R6G) is collected with graphene/Ag nanoholes (GE/AgNHs) structures as SERS substrates, and the corresponding normalized intensity is also calculated and discussed in details. The relative standard deviation (RSD) is reduced from 25% (Raman intensity) to 12% (normalized intensity). The quantification of R6G is demonstrated down to the detection limit of 10−15 M.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Surface-enhanced Raman scattering (SERS) has been widely used to detect trace species, and even single molecule studies in many fields, such as chemical, life science, and medical applications [1–4]. The enhancement of the SERS originates mainly form highly concentrated electromagnetic hotspots, induced by plasmon excitation in metal nanostructures. It is largely relying on the optimized metal nanostructures using localized surface plasma resonance [5,6]. However, due to random distribution of the hotspots, molecular adsorption on the surface of metal surface, fluctuation of optical setup, it is difficult to obtain reliable analysis results [3]. Thus, SERS suffers from poor reproducibility, and quantitative SERS as a powerful analytical tool in practical applications is still unsolved.
A great number of studies and techniques have been focused on the improvement of the reproducibility of SERS substrates, for example template techniques [7,8], electron beam lithography [9], nanoimprint lithography [10,11], and self-assembly of metal nanoparticles [12–14]. With the development of nano fabrication technology and new nano materials, different SERS substrates with different metal structures and graphene/carbon nanotubes have been improved [6,13,15–18]. However, some problems cannot be solved. Firstly, the enhancement property still has fluctuation for different SERS substrates samples at different times and different batches, leading to the uncertainty in the absolute Raman intensity [19]. Secondly, the inevitable fluctuation of molecules on the metal surface also leads to unstable signal intensity. Some core-shell structures are developed to resolve such problem [20,21]. Meanwhile, internal standard method is utilized to solve the above disadvantages. There are three ways to complete internal standard method. One is that an internal molecule is embedded into SERS substrates by a core-molecule-shell structure [20]. But the preparation is very complicated, and the selection of the internal standard specie is limited by the preparation method. The other one is that an internal molecule is directly added into the analyte molecule [22]. But, the internal standard species would affect and even replace the surface adsorption site of the analyte molecule. Maybe a better choice of the internal standard specie is an isotopically substituted form of the target analyte itself [19]. However, such internal standard specie is very difficult to obtain and the cost is also high. In addition, the latest reported one is that an internal standard specie is indeed in the SERS substrate itself. The ratio between the Raman intensity of the analyte molecule and the internal standard specie can be used for quantification. A method based on the intensity of the surface plasmon enhanced Rayleigh band that originates from the amplified spontaneous emission of the laser is proposed to complete quantitative SERS analysis [23]. Moreover, a graphene/Au nanoparticles substrate for SERS quantification is reported [3], and the feasibility of quantification of crystal violet (CV) molecules in aqueous with concentration ranging from 10−8 to 10−5 M. To this end, an investigation of an integration of period metal nanostructure and an internal standard material in SERS substrates is an important task.
Herein, we report a graphene/Ag nanoholes (GE/AgNHs) substrate for analyte quantification. Ag nanoholes structure has uniform distribution with 300 nm period in a Ag film, and it brings high Raman enhancement. Graphene has three functions. Firstly, it is helpful to quench the fluorescence and provide clean Raman signal due to the separation of molecules from a metal. Secondly, it is benefit to protect the oxidation of metal nanostructure. Thirdly, graphene is naturally an internal standard to normalize the Raman intensity of analytes. We also investigate the quantification of our samples with rhodamine 6G (R6G) as analyte molecule.
2. Structures
Figure 1(a) illustrates the scheme of the GE/AgNHs SERS substrate. Ag film with thickness of 110 nm is deposited on SiO2 substrate. There are three steps to synthesis the samples. First, Ag nanoholes are prepared by surface plasmon (SP) lithography technology [24–27]. The width and depth of hole can be tuned by control of etching process. Secondly, large-area graphene is prepared on Cu foil via chemical vapor deposition (CVD) [28]. Finally, graphene is transferred onto the target substrate with a wet transfer method [29]. The scanning electron microscope (SEM) image of the prepared Ag nanoholes structure is shown in Figs. 1(b) and 1(c). We can see the gap of the holes is ~50 nm, and the period of holes is ~300 nm.
Fig. 1 (a) The schematic image of GE/AgNHs, area A is Ag film, area B is Ag nanoholes, area C is the transferred graphene. The amplified area B is also shown, the period of Ag nanoholes is 300 nm, the height of holes is 70 nm. (b) The SEM image of area B, and (c) the enlarged SEM image of area B. (d) The Raman signals of graphene on SiO2 (black line) and Ag nanoholes (red line), the corresponding optical images are inserted. (e) Electrical field distribution at the top surface of GE/AgNHs, with FDTD.
The Raman spectra were collected with a laser confocal Raman spectrometer (Horiba JY LabRAM HR Evolution), equipped with a 50 × objective lens (numerical aperture of 0.75, work distance of 0.37 mm), using a green laser (λ = 532 nm) with a ~1 μm laser spot diameter. The power of the laser is 5 mW (50 mW with a 10% neutral density filter) to avoid sample heating and photoinduced damages. An integration time of 2 s was used in the measurements. The Ag nanoholes ensure that the Raman enhancement is still dominated by the localized electromagnetic enhancement. Figure 1(d) illuminates the Raman spectra and the optical microscope images of graphene on SiO2 and our Ag nanoholes. We can observe a weak D peak and a good macroscopic continuity on SiO2 substrate, showing less defects and high quality [30]. The ratio between G peak and 2D peak is 0.602, which means the graphene is mono-layer [31]. Compared with graphene on SiO2 substrate, graphene on Ag nanoholes shows an enlarged D peak, meaning that there are some defects during the transfer process and electromagnetic enhancement by Ag nanoholes [32,33]. The enhancement of D peak is mainly by the doping effect and Ag surface, leading to an increase of defects such as wrinkles, cracks or residues on graphene. In addition, the intensity of G and 2D peaks has an obvious enhancement. It is caused by the electromagnetic enhancement (EM) of Ag nanoholes and the plasmonic coupling induced by the interaction between AgNHs and GE, where hot electrons can be injected into GE layer [29]. The enhancement degree is affected by the metal nanostructures.
In order to see clearly the EM effect, the spatial distribution of the electromagnetic field intensity for GE/AgNHs is simulated with the FDTD (Finite-difference time-domain) method, using periodic boundary conditions, show in Fig. 1(e). The simulation is carried out in vacuum (nmedium = 1.0). The wavelength of incident light is 532 nm and the incident field intensity is E0 = 1 V/m. The substrate was laid on the X-Y plane and the incident electric field with X-polarization propagated along the -Z direction. The calculated local electric fields have reached the maximum of 36.2 V/m, and the corresponding calculated enhancement factor is ~1.7 × 106.
3. Mechanism of quantification
Figures 2(a) and 2(b) show the scheme of quantitative analysis using GE/AgNHs structures. Graphene is naturally an internal standard for the normalization of the Raman intensity of analyte molecules. By simultaneously collecting the Raman signals from the analyte (IAnalyte) and graphene (IGE), the different enhancement of analyte molecules at different locations can be calibrated with 2D peak of graphene. A relative intensity parameter k (the ratio of IAnalyte and IGE) is calculated for further discussion. We chose 2D peak of graphene because the position of 2D peak is far away from the typical frequency window of our analyte molecule R6G, and its intensity is comparatively strong.
Fig. 2 (a) Scheme of quantitative analysis using GE/AgNHs structures; (b) a representative Raman signal of R6G on GE/AgNHs sample, one Raman peak of R6G is labelled with red pentagram, while that of graphene labelled with blue star. The parameter k is used to show the normalized Raman intensity. (c) Several Raman signals of different concentration in the range of 10−15 M to 10−10 M. Raman intensity of graphene on SiO2 is inset with a black line. (d) The Raman intensity and normalized intensity (2D peak as internal standard) change with R6G concentration; the linear fit of k is shown in red dot line, with R2 of 97%.
Figure 2(c) illustrates several random representative Raman intensities of R6G with concentration ranging from 10−15 to 10−10 M on our SERS samples, and the Raman peak at ~2682 cm−1 (2D peak of graphene) is clearly observed. The Raman intensity at 613 cm−1 is chose for further analysis. To quantitatively illustrate the relationship between the Raman intensity and the parameter k, shown in Fig. 2(d), the parameter k is almost linear with the concentration, with R2 of 97%.
The Raman intensity of the peak at 613 cm−1 does not orderly change. There are some reasons. When we took experiments, the focus plane would not exactly be the same position, due to handmade process. In additions, the non-uniformity of enhancement hotspots is another factor. However, the two factors mentioned above will also affect the Raman intensity of graphene. When we take calculation with the Raman intensity of 613 cm−1 and graphene, the effects induced by the two factors would be largely reduced, even eliminated theoretically.
4. Raman mapping measurement
In addition to the random intensity, the signal reproducibility across a SERS substrate is also an important factor. In order to further weaken random measurement errors, Raman mapping in areas of 10 μm by 10 μm is carried on. The averaged Raman intensity of R6G with concentration of 10−12 M is shown in Fig. 3(a). We can clearly see Raman peaks of R6G and 2D of graphene. The Raman mapping image is represented in Fig. 3(b). The Raman signals of R6G and graphene fluctuated simultaneously. We calculate the ratio k for each Raman spectrum within the mapping area, and then plot the probability density function. The probability density function of the ratio k (I@613/I@2D) is shown in Fig. 3(c), in which a lognormal distribution is shown to be a good fit, with the mean value of 1.43. The relative standard deviation (RSD) of the peak intensity at 613 cm−1 and 773 cm−1 is 25% (Fig. 3(d1)) and 27% (Fig. 3(e1)), respectively. While the RSD of parameter k is reduced to 12% (Fig. 3(d2)) and 17% (Fig. 3(e2)), respectively, which implies that such internal standard method is potential for reliable quantitative analysis. Moreover, compared with other AuNPs via seed-mediated growth method [23], our sample has better uniformity.
Fig. 3 (a) The averaged Raman intensity of R6G with concentration of 10−12 M; (b) Raman mapping data of R6G; (c) probability density function of the normalized intensity (data at 613 cm−1 used), showing a good lognormal distribution with a lognormal median at k = 1.43. The corresponding RSD of (d1) Raman intensity at 613 cm−1 and (d2) the calculated parameter k. The corresponding RSD of (e1) Raman intensity at 773 cm−1 and (e2) the calculated parameter k.
In addition, in order to demonstrate reliable SERS quantification, we further to take Raman mapping measurement for R6G with other different concentration. Figure 4(a) illuminates the averaged Raman spectra of R6G with concentration ranging from 10−10 to 10−15 M. Figure 4(b) shows some representative Raman mapping spectra of R6G with concentration of 10−11 M. Shown in Figs. 4(c) and 4(d), it can be see that the intensity of R6G increased with the increase of concentration, except data of 10−11 and 10−12 M, probably due to different focus status and non-uniform analyte molecule absorption characteristics on different samples. Fortunately, the calculated averaged k indeed increased with the increase of concentration, exhibiting a good linear relationship between k and R6G concentration. The R2 of linear fit is 99% and 97% for data of 613 cm−1 and 773 cm−1, respectively. Compared with graphene/AuNPs composites [3], the quantification of analyte molecules is demonstrated down to the lower detection limit of 10−15 M.
Fig. 4 (a) The averaged Raman intensity of R6G with different concentration in the range of 10−15 M to 10−10 M. (b) Some representative Raman mapping spectra of R6G with concentration of 10−11 M. The Raman intensity and normalized intensity k change with R6G concentration at (c) 613 cm−1 and (d) 773 cm−1. Red dots are the averaged k with a linear fit, while black squares are the averaged Raman intensity. The corresponding error bar is also shown.
Moreover, we also noticed that the Raman intensity of graphene fluctuated from Fig. 4(a). There could be two factors. One is the different enhancement induced by different electromagnetic enhancement of Ag nanoholes. The other is due to chemical reaction between graphene and R6G with different concentration.
5. Conclusion
In summary, a graphene/Ag nanoholes SERS substrate has been developed for quantification analysis. Ag nanoholes play a role of electromagnetic enhancement, while graphene plays roles of an internal standard, a platform for adsorption of analyte molecule, and a protection layer for oxidation of metal surface. The reliability of quantification of R6G with concentration ranging from 10−15 to 10−10 M is investigated. Thus, such method has great potential for quantitative SERS.
Funding
National Natural Science Foundation of China (No. 61376121); Natural Science Foundation of Chongqing (No.CSTC2015JCYJBX 0034); the Fundamental Research Funds for the Central Universities (CQU2018CDHB1A07).
Acknowledgments
We would like to thank Analysis and Test Center of Chongqing University. We also thank Mr. X. N. Gong for Raman measurement help.
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