Facial Synthesis of Carrageenan / Reduced Graphene Oxide / Ag Composite as Efficient SERS Platform

In this paper, we reported the preparation of carrageenan/reduced graphene oxide/Ag composite (CA-RGO-Ag) by a wet chemical method at room temperature using carrageenan, graphene oxide and silver nitrate as starting materials. As-prepared composite was characterized by UV-vis spectroscopy, Raman spectroscopy, FTIR, SEM, EDX and XRD. Results showed that the reduction of graphene oxide (GO) and silver nitrate was achieved simultaneously by addition of NaBH4. Surface-enhanced Raman scattering study showed that the obtained composite give an intensive and enhanced Raman scattering when Rhodamine B was used as a probing molecule.


Introduction
Surface enhanced Raman scattering (SERS) is a powerful analytical tool that can provide ultrasensitive detection of chemical and biological molecules down to the single molecular level, which has been used in various fields of chemistry, material science and biophysics 1 . There are two SERS mechanisms that are generally accepted: electromagnetic mechanism (EM) and chemical mechanism (CM) [2][3][4] . EM is based on the enhancement of the local electromagnetic field while the CM is based on charge transfer between absorbed molecules and substrate surface. Up to now, many surfaces have been successfully investigated as SERS active substrates such as Ag, Au, Cu, Na, Li, Pd, Pt, the best performances for detection purposes have been recorded in the case of Ag and Au nanoparticles [5][6][7][8][9][10][11][12][13][14][15][16] .
Graphene, a two-dimensional monolayer sheet of hexagonal carbon atoms, has attracted great attention because of its unique structure and exceptional physiochemical properties [17][18][19][20] . It has previously been reported to play a role as a Raman enhancement substrate because target molecules can easily form a unified orientation on it due to the flatness of the graphene surface 21,22 . Moreover, the interconnected sp 2 network of graphene gives it the ability to undergo π−π stacking with aromatic molecules 23,24 . However, most of the graphene used for preparing SERS substrate are in its reduced form from graphene oxide (GO) prepared by the oxidation of graphite. The reduced graphene oxide (RGO) is prone to irreversible spontaneous agglomeration, which highly limits its applications and performances 25,26 . Therefore, surface functionalization is considered as an effective way to solve this problem [27][28][29][30][31] . In this report, we prepared an enhanced sensitivity for SERS detection using carrageenan functionalized RGO-Ag (CA-RGO-Ag) composite. The prepared composite was characterized by a series of techniques and its SERS property was evaluated by the detection of Rhodamine B (RhB) and adenine.

Synthesis of CG/RGO/Ag composite
Graphene oxide was prepared using modified Hummer's method 32,33 . In a typical procedure, 125 ml of concentrated sulfuric acid was taken into a flask filled with graphite powder (5 g) followed by the addition of KMnO 4 (17.5 g) slowly at 0 °C. The mixture was stirred for 3 h at 35 °C and then diluted by water at 0 °C. After that, H 2 O 2 (30 vol.% in water) was added into mixture until the bubbling of the gas was completed. The graphene oxide (GO) powder was collected by centrifugation of the solution and subsequently dried under vacuum at 80°C for 24 h.
To synthesize CG/RGO/Ag composite, 0.5 mg carrageenan was dissolved into 50 mL water by 30 min sonication to form a brown solution. 10 mL GO dispersion (1 mg/mL) was a Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing Botanical Garden, Memorial Sun Yat-Sen, Nanjing 210014, China b Department of Physics and Materials Science, City University of Hong Kong, Hong Kong added into carrageenan solution for 1 h stirring. After that, a certain amount of AgNO 3 solution (50 mM) was added into the mixture for another hour stirring. Then, 1 mL NaBH 4 solution (0.5 M) was added to the above mixture dropwise. The color of the mixture turned from brown to black. After 30 min stirring, the composite was centrifuged, washed and dried at 80°C for 24 h. (denoted as CG/RGO/Ag-1, CG/RGO/ Ag-2, CG/RGO/Ag-3, CG/RGO/Ag-4, CG/RGO/Ag-5 and CG/RGO/Ag-6 for the weight ratios of GO and AgNO 3 set as 1:5, 1:10, 1:20, 1:30, 1:40 and 1:50, respectively). CG/RGO and RGO/Ag sample also synthesized using a similar method except addition of AgNO 3 and carrageenan, respectively.

Characterizations
Surface morphology of samples were analyzed by scanning electron microscope (SEM, S-4700, HITACHI). FTIR spectra were obtained using a Nicolet 8700 FTIR spectrometer (Thermo Scientific Instrument). X-ray diffraction patterns were collected from 10° to 60° in 2θ by a XRD with Cu Kα radiation (D8-Advanced, Bruker). Raman analysis was carried out at room temperature using a Raman spectroscope (Renishaw InVia, UK) with a 514 nm laser light. The optical characterizations were obtained by UV-vis spectrophotometer in the wavelength range from 200 to 500 nm.

SERS study
For SERS study, 20 μL of CA-RGO-Ag dispersion was dropped onto the glass substrate and dried at room temperature. Then, 20 μL of 1.0 × 10 ─4 M ethanol solution was dropped onto the CA-RGO-Ag films and dried at room temperature, and SERS spectra were recorded, respectively.

Results and Discussion
UV-vis spectroscopy was used for confirming the formation of Ag nanoparticles (NPs) and reduction of GO. Figure  1A displays the UV-vis spectra of GO and CA-RGO-Ag composite. It can be seen that the spectrum of GO exhibits two characteristic absorption peaks at 227 nm and 315 nm, corresponding to π─π* transitions of aromatic C─C bonds and n─π* transitions of C=O bonds, respectively 34 . However, the peak at 227 nm of GO dispersion gradually red-shifts to 262 nm and the shoulder absorption peak at 316 nm disappear in the spectrum of CA-RGO-Ag-6, which indicates the GO has been reduced by NaBH 4 . Furthermore, a new broad absorption peak at 425 nm is observed in the spectrum of CA-RGO-Ag-6, which could assign to the surface plasmon resonance absorption band of Ag nanoparticles, suggesting the successful formation of Ag NPs. Figure 1B displays the FTIR spectra of GO and CA-RGO-Ag-6. As expected, the spectrum of GO exhibits signals at 3432, 1638, 1156 and 1038 cm ─1 corresponding to the ─OH vibration stretching, carboxyl C=O, epoxy C─O and alkoxy C─O, respectively 35 . It can be seen that these peaks show a relatively lower intensity or even vanished in the spectrum of CA-RGO-Ag-6, further confirm the reduction of GO. Moreover, the spectrum of CA-RGO-Ag-6 also shows peaks at 1225, 915 and 852 cm ─1 , which could be assigned as the characteristic peaks of carrageenan, suggesting the successful surface functionalization of carrageenan 36,37 .
Raman spectroscopy was also used for determining the structural changes of the as-prepared samples. The Raman spectra of graphite, GO and CA-RGO-Ag-6 are shown in Figure 2. As expected, a prominent peak at 1580 cm ─1 is observed in graphite sample, which corresponds to the firstorder scattering of the E 2g . The spectrum of GO displays two characteristic main peaks for D band at 1330 cm ─1 and G bands at 1585 cm ─1 . The G band is attributed to all sp 2 carbon forms and provides information on the in-plane vibration of sp 2 bonded carbon atoms while the D band suggests the presence of sp 3 defects 38 . The intensity ratio between D band and G band (I D /I G ) increases from 0.91 in GO to 1.1 in CA-RGO-Ag-6 is due to the decrease of average size of sp 2 domains by reduction of GO 39 .
The morphology of as-prepared samples were examined by SEM. Figure 3A Figure 3B) exhibits a much larger size. For CA-RGO-Ag samples, the RGO sheets are embedded into carrageenan gel. On the surface, we could observe a uniform distribution of single Ag nanoparticles when a higher AgNO 3 content was introduced during the preparation. The average size of the Ag NPs can be calculated to be 22 nm (for CA-RGO-Ag-5) based on 100 individual Ag NP size measurements. EDX analysis has been used to obtain elements information about CA-RGO-Ag-6 ( Figure  4). The spectrum presents the only existence of C, O (belong to RGO), S (belongs to carrageenan) and Ag (belongs to Ag NPs), indicating the successful formation of composite with high purity.
The powder X-ray diffraction of the samples are shown in Figure 5. It is clear that the XRD pattern has peaks at 25.29°, which correspond to (002) crystal planes of graphite, indicating the presence of layered RGO sheets structure. RhB was chosen as the model molecule to examine the performance of CA-RGO-Ag SERS substrates. Figure 6 shows a set of the SERS spectra of 1×10 ─4 RhB on glass slide, Facial Synthesis of Carrageenan/Reduced Graphene Oxide/Ag Composite as Efficient SERS Platform   RGO-Ag and CA-RGO-Ag-3 substrates. It can be found that the 10 ─4 M RhB molecules alone (a) almost have no Raman signals. However, the spectra collected from RGO-Ag and CA-RGO-Ag-3 substrates all show distinct peaks at 624 (C─C─C ring in plane bend), 760 (C─H out of plane bend), 829 (C─C), 938 (C─H stretch), 1076 (β (CH)), 1191 (C─H in plane bend), 1277, 1362, 1502 (Arom C─H stretch), 1567, 1650 (Arom C─C stretch) and 1596 cm ─1 (C─H stretch) [40][41][42] . Compared SERS signals on RGO-Ag (b) with the ones on CA-RGO-Ag-2 (c), it can be found the SERS signal on CA-RGO-Ag-3 is stronger than the one on RGO-Ag, which could attract to both electromagnetic and chemical factors. As observed in the SEM characterizations, the introduction of carrageenan effectively prevents the formation of large cluster Ag NPs on the RGO sheets. The uniform distributed small Ag NPs play the role of electromagnetic hot spots that have intense local electromagnetic fields, resulting in highly efficient Raman scattering 43 . On the other hand, the carrageenan surface functionalization could prevent the aggregation of RGO sheets, which could adsorb and concentrate the target molecules and magnify the SERS signal 44 .
In order to optimize the performance, the SERS spectra were collected from the CA-RGO-Ag substrate with different composition. Figure 7    all the spectra, the SERS spectrum collected from CA-RGO-Ag-5 shows the highest enhancement performance, which could ascribe to the uniformity of Ag NPs distribution and optimized Ag NPs size adjusted by the interaction between Ag + and GO during the preparation process. Therefore, the  weight ratio of GO and AgNO 3 for fabricating CA-RGO-Ag SERS substrate was set as 1: 40.
The practical application of CA-RGO-Ag SERS substrate was tested by detection of adenine, a kind of nucleotide based for DNA sequencing. The SERS spectra of adenine (1 × 10 ─4 M) on the RGO-Ag and CA-RGO-Ag-5 are shown in Figure 8. It can be found that four characteristic peaks are pronounced at 730, 1332, 1444 and 1587 cm ─1 , which are corresponded to the purine ring breathing mode, the C-N stretching mode, C-N in plane stretching mode and sciss NH 2 , respectively 45,46 . Moreover, three peaks at 655, 687 and 1054 cm ─1 were also observed when using CA-RGO-Ag-5 as SERS substrate, indicating the proposed substrate exhibits an excellent SERS activity towards detection of adenine.

Conclusion
In conclusion, CA-RGO-Ag composite has been prepared via a facial wet chemical route using NaBH 4 as reducing agent. The as-prepared composite exhibits strong plasmonic resonances and can be used as a SERS substrate. The SERS activity of the CA-RGO-Ag substrate was optimized by varying the weight ratio of AgNO 3 and GO in the preparation process. The improved activity can be attributed to the uniform distributed small Ag NPs, which provides intense local electromagnetic fields, resulting in highly efficient Raman scattering. On the other hand, the carrageenan functionalized RGO sheets adsorb and concentrate the target molecules and magnify the SERS signal.