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Assembly of Graphene Oxide and Au0.7Ag0.3 Alloy Nanoparticles on SiO2: A New Raman Substrate with Ultrahigh Signal-to-Background Ratio Peng Chen,†,‡ Zongyou Yin,† Xiao Huang,† Shixin Wu,† Bo Liedberg,‡,§ and Hua Zhang*,†,‡ †
School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 Center for Biomimetic Sensor Science, 50 Nanyang Drive, Research Techno Plaza sixth storey, Singapore 637553 § Department of Physics, Chemistry and Biology (IFM), Link€oping University, SE581 83 Link€oping, Sweden ‡
bS Supporting Information ABSTRACT: Resonance Raman spectroscopy (RRS) often suffers from the large fluorescence background which obscures the much weaker Raman scattering. To address this fundamental problem, a novel Raman substrate has been fabricated by adsorption of Au0.7Ag0.3 alloy nanoparticles (NPs) on a graphene oxide (GO) coated SiO2 surface, which offers both excellent Raman enhancement and fluorescence quenching. Our experimental data reveal that a Raman to fluorescence background intensity ratio of 1.6 can be obtained for a highly fluorescent dye like Alexa fluor 488. Moreover, we demonstrate that the Raman enhancement mainly originates from the Au0.7Ag0.3 alloy NPs and that the fluorescence quenching mainly arises from the underlying functionalized GO (FGO) substrate.
’ INTRODUCTION Graphene,13 a single layer of hexagonally arranged sp2 carbon atoms, has attracted tremendous interest in both fundamental and applied research because of its unique electrical,4,5 physical,6 optical,7 and mechanical properties.8 Importantly, its excellent fluorescence quenching properties recently have been used for graphene-based biosensors7 and for reducing the fluorescence background in resonance Raman scattering spectra.9 Resonance Raman spectroscopy (RRS) is a powerful technique used to characterize the chemical structure of molecules at low concentration.1012 However, RRS often suffers from the high fluorescence background1315 because of the relative small Raman cross section (1022 cm2) compared to that of fluorescence (1016 cm2).12,16 Therefore, the signal-to-background (S/B) ratio is often too low to resolve the Raman characteristic peaks of organic molecules, especially for highly fluorescent molecules/ dyes.12,15 Traditional attempts to improve the S/B ratio involve surface-enhanced Raman scattering (SERS), that is, the use of noble metal nanostructures as substrates to enhance the Raman scattering.1721 Very recently, a brilliant alternative has been suggested where the strong fluorescence of target molecules is quenched by attaching them to graphene.22 Although the aforementioned methods can increase the S/B ratio, there is still much room for improvement if one considers that the cross section value of fluorescence is 6 orders higher than that of Raman scattering.12,16 Herein, we report a novel method to increase the S/B ratio by combining the advantageous properties r 2011 American Chemical Society
of graphene oxide (GO) and alloy Au0.7Ag0.3 nanoparticles (NPs) on SiO2. A highly fluorescent dye, Alexa fluor 488, is used as Raman tag to demonstrate the capability of this novel Raman substrate. Although recently graphene/metal nanostructure composites have been used to enhance the D and G bands of graphene,23,24 to the best of our knowledge, this is the first study reporting the improvement of the S/B ratio of highly fluorescent molecules by integrating Raman enhancement from noble metal NPs and fluorescence quenching from graphene.
’ RESULTS AND DISCUSSION Scheme 1 shows the assembly process of GO and Au0.7Ag0.3 nanoparticles (NPs) on SiO2. GO was synthesized by a modified Hummers method.25 After GO was activated by NaOH solution to introduce ionized carboxylic groups (COO),26 it was adsorbed onto 3-aminopropyltriethoxysilane (APTES)-modified SiO2 surface13,27 to form GO films.25,28,29 The GO films with COO groups were then functionalized with ethylene diamine (NH2CH2CH2NH2),30 which yielded the functionalized graphene oxide terminated with amine groups (NH2 ). The negatively charged alloy Au0.7Ag0.3 NPs, synthesized by simultaneous reduction of HAuCl4 and AgNO3 with trisodium citrate,31 were assembled onto the amine-functionalized GO (FGO) Received: September 2, 2011 Revised: October 26, 2011 Published: October 28, 2011 24080
dx.doi.org/10.1021/jp208486m | J. Phys. Chem. C 2011, 115, 24080–24084
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Scheme 1. Assembly Process of GO and Au0.7Ag0.3 NPs on an APTES-Modified SiO2 Substrate
Figure 1. (a) FESEM image of GO adsorbed on APTES-modified SiO2 substrate. (b) Raman spectra of graphite flakes, GO, activated GO, and amine-functionalized GO (FGO). (c) TEM image of Au0.7Ag0.3 NPs. (d) UVvis spectrum of Au0.7Ag0.3 NP solution.
surface through the electrostatic interaction. Finally, the novel Raman substrate, referred to as SiO2/FGO/AuAg NP, was obtained (Scheme 1). The field emission scanning electron microscope (FESEM) image in Figure 1a clearly shows that the activated GO sheets were successfully adsorbed on the APTES-modified SiO2 substrate, where most GO sheets are single-layered with thickness of ∼0.9 nm (Figure S1 in the Supporting Information, SI), which is consistent with our previous results.25,28,29 Figure 1b shows the Raman spectra of graphite and GO with different functionalization. The mechanically cleaved graphite flakes give a sharp peak corresponding to the first-order scattering of E2g mode (G band)32 at 1584 cm1 and to a second-order zone-boundary phonon peak (2D band)33 at 2700 cm1. The characteristic peak (D band) at 1350 cm1, arising from zone-boundary phonons, was not observed in the defect-free graphite.34 After chemical oxidation of graphite, many oxygen-containing functional groups were introduced,35 which changed the chemical and electronic properties of graphite, as evidenced by the increased D band and decreased 2D band in GO (green line in Figure 1b). In addition, the G band blue-shifted to 1600 cm1, which is attributed to the formation of alternating single and double carbon bonds induced by the oxygen-containing functional groups.36 After GO was activated with NaOH, an increase of D/G ratio from 0.69 to 0.79
was observed (blue line in Figure 1b), since NaOH not only activated the carboxylic groups26 but also removed some oxygencontaining groups from the GO surface.37,38 The reaction of diamine with carboxylic groups on GO surface, referred to as amine-functionalized GO (FGO), did not change the D/G ratio (red curve in Figure 1b), indicating that the coupling reaction only occurred in the carboxylic groups. The relative constant peak position of G band also revealed that the diamine coupling did not induce any changes to the GO basal plane. AuAg alloy NPs were synthesized through the simultaneous reduction of HAuCl4 and AgNO3 by trisodium citrate.31 The plasmonic response of the alloy NPs can be tuned continuously from the pure Ag NPs (∼400 nm) to the pure Au NPs (∼520 nm) by varying the ratio of Au and Ag.31 Transmission electron microscope (TEM) image of the AuAg alloy NPs is shown in Figure 1c. Figure 1d shows the UVvis absorption spectrum of the synthesized alloy NPs with the plasmonic resonance peak appearing at 489 nm. The composition of the alloy NP was examined by the energy-dispersive X-ray spectroscopy (EDX), and the obtained ratio of Ag to Au is ∼3:7, that is, Au0.7Ag0.3 (Figure S2 in the SI), which is consistent with the previous prediction based on the equation of λAuAg = 412 + 1.13χAu, where χAu is the percentage of Au atoms.31 The FESEM image of SiO2/FGO/AuAg NP substrate is shown in Figure 2a. The density of Au0.7Ag0.3 NPs is estimated to be 8.87/μm2. In our experiment, three other kinds of substrates, that is, APTES-modified SiO2, SiO2/FGO, and SiO2/AuAg NP (see the substrate preparation in Experimental Section for details), were used for the comparative studies. The density of Au0.7Ag0.3 NPs adsorbed on APTES-modified SiO2 substrate, that is, SiO2/AuAg NP, is estimated to be 8.83/μm2 (Figure 2b), which is similar to that on SiO2/FGO/AuAg NP substrate. Because of the short incubation time (∼30 min) of substrates in the Au0.7Ag0.3 NP suspension used in our experiments, some free amine groups still existed on both SiO2/FGO/AuAg NP (Figure 2a) and SiO2/AuAg NP surfaces (Figure 2b), which could be used for immobilization of dye molecules. After a drop of sodium bicarbonate buffer solution containing 1 μM Alexa fluor 488 was dropped onto the aforementioned four kinds of substrates which were incubated for 10 min, the samples were carefully rinsed with Milli-Q water. Resonance Raman spectroscopy (RRS) was conducted to evaluate both the Raman scattering and the fluorescence of Alexa fluor 488 on these substrates. The absorption and fluorescence spectra, chemical structure of Alexa fluor 488, and its reaction with FGO are shown in Figure S3 in the SI. As known, the SiO2 substrate has no effect on the fluorescence intensity and Raman scattering. With excitation of 488 nm laser, the Alexa fluor 488 dye is highly fluorescent, and the strong fluorescence obscures the weak Raman scattering as shown in the red 24081
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Figure 2. (a) FESEM image of SiO2/FGO/AuAg NP substrate. Inset: High magnification image. (b) FESEM image of SiO2/AuAg NP substrate.
Figure 3. (a) Raman spectra of Alexa fluor 488 dye adsorbed on APTES-modified SiO2 (red curve), SiO2/AuAg NP (blue curve), SiO2/FGO/ AuAg NP (black curve), and SiO2/FGO (green curve). The peak of Si at 520 cm1 is used as reference. Characteristic peaks of Alexa fluor 488 in blue curve are indicated by stars. The spectrum intensity of Alexa fluor 488 on SiO2 is divided by a factor of 5. Inset: Magnified Raman spectrum of Alexa fluor 488 on SiO2/FGO in the dotted area of green curve. (b) Plot of S/B ratio of Alexa fluor 488 on different substrates: SiO2 (106), SiO2/AuAg NP (0.039), SiO2/FGO (0.12), SiO2/FGO/AuAg NP (1.6).
curve in Figure 3a. This is due to the fact that the Raman scattering (cross sections of 1022 cm2) to fluorescence (cross sections of 1016 cm2) ratio is ∼106, which even made the strong characteristic peak of Si (∼520 cm1) unobserved. However, the SiO2/AuAg NP substrate gave rise to weak Raman signals arising from the enhancement by the magnified local electromagnetic field near the NPs (blue curve in Figure 3a). Although the strong fluorescence still made the Raman spectra very noisy, at least some Raman characteristic peaks could be identified as indicated by the stars. In contrast to the aforementioned strategy for exclusively enhancing the Raman scattering by using the SiO2/AuAg NP substrate, the SiO2/FGO substrate proved to be even better to increase the signal-to-background (S/B) ratio as illustrated by the green curve in Figure 3a. The SiO2/FGO substrate almost eliminated the fluorescence background from the Raman spectrum of Alexa fluor 488. This observation is consistent with the previous report on the fluorescence quenching by using graphene.9 As seen from the inset of the Raman spectrum of Alexa dye on SiO2/FGO in Figure 3a, the Raman peaks of the dye molecules in the 15001700 cm1 region are clearly observed. Raman peaks of D band around 1343 cm1
and G band around 1595 cm1 are characteristic signatures of graphene oxide. Importantly, the Alexa fluor 488 dye on SiO2/FGO/AuAg NP substrate resulted in a strong and well-resolved Raman signal (the black curve in Figure 3a), where the most dominant peak at 1647 cm1, attributed to the stretching of CdC bonds in the fused benzene ring,11,21 was used for calculation of the S/B ratio in Figure 3b. In addition to the Raman enhancement, the fluorescence intensity of Alexa dye also decreased significantly compared to that on SiO2 (the red curve in Figure 3a). Figure 3b summarizes the S/B ratio of the aforementioned four substrates. The calculation of S/B ratio is illustrated in Figure S4 of the SI by using SiO2/FGO/AuAg NP as an example. Compared to SiO2, the higher S/B ratio obtained on SiO2/AuAg NP and SiO2/ FGO clearly demonstrated that the Raman enhancement and fluorescence quenching arose from Au0.7Ag0.3 NPs and FGO, respectively. The observed S/B ratio of 1.6 is approximately 106 higher than that on SiO2, indicating a successful integration of Au0.7Ag0.3 NPs and FGO to form the novel Raman substrate, resulting in the synergistic effect between the strong Raman enhancement and the strong fluorescence quenching which arose from Au0.7Ag0.3 NPs and FGO, respectively. 24082
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The Journal of Physical Chemistry C The following working principle is proposed for the SiO2/ FGO/AuAg NP assembly. The dye molecules are expected to be adsorbed on the FGO surface rather than on the Au0.7Ag0.3 NPs because of the covalent bonding formed between the dye molecule and the amine-functionalized GO (FGO) (Figure S3C of the SI) and the electrostatic repulsion between the negatively charged dye molecule and the Au0.7Ag0.3 NP. With illumination by the 488 nm laser, the fluorescence from the dye molecules is quenched by FGO, while the Raman signal of the dye molecules in close proximity to the Au0.7Ag0.3 NPs is enhanced. The mechanism of Raman enhancement in the SiO2/FGO/AuAg NP substrate is dominated by the electromagnetic enhancement from the Au0.7Ag0.3 NPs.13 Although graphene was reported to provide some chemical enhancement9,39,40 of Raman signals because of the charge transfer, the effect is much less than that of electromagnetic enhancement from noble metals.40,41 Since the distance between dye molecules and Au0.7Ag0.3 NPs is quite large because of the electrostatic repulsion, the fluorescence quenching from Au0.7Ag0.3 NPs on dyes can never be high.42 Therefore, we believe that the fluorescence quenching of dye molecules on SiO2/FGO/AuAg NP mainly arises from FGO possibly because of the resonance energy transfer43 or the charge transfer44 between dye molecules and FGO.
’ CONCLUSIONS In summary, a novel SiO2/FGO/AuAg NP Raman substrate has been successfully fabricated by immobilization of the negatively charged Au0.7Ag0.3 NPs onto the positively charged FGO film on SiO2 through the electrostatic interaction. This novel substrate inherits the excellent fluorescence quenching property from FGO and simultaneously integrates the Raman enhancement property from the Au0.7Ag0.3 NPs. By using the highly fluorescent dye Alexa fluor 488 as an example, SiO2/FGO/AuAg NP as a Raman substrate gave 6 orders of magnitude improvement in the signal-to-background ratio compared to the SiO2 substrate. This novel SiO2/FGO/AuAg NP Raman substrate not only solves the problem of high fluorescence background in resonance Raman scattering but also opens up a new avenue for Raman characterization of molecules which are not only the commonly used Raman dyes but are also the highly fluorescent molecules (e.g., Alexa fluor 488 used here). This achievement is significant given the fact that most molecules in the real world are not Raman dyes. ’ EXPERIMENTAL SECTION Synthesis and Activation of Graphene Oxide. Graphene oxide (GO) was prepared by the modified Hummers method.25,45 The obtained GO powder (10 mg) was dissolved in 50 mL of 3 M NaOH aqueous solution and then was kept for 30 min to ionize the carboxylic groups on GO surface.26 The obtained activated GO was washed with Milli-Q water for several times until the pH value decreased to 8. Synthesis and Characterization of Au0.7Ag0.3 Alloy Nanoparticles. AuAg alloy nanoparticles (NPs) were obtained by simultaneous reduction of the mixture of HAuCl4 and AgNO3 in trisodium citrate solution.31 Briefly, 875 μL of 10 mM HAuCl4 aqueous solution was added into 50 mL Milli-Q water with refluxing. Then, 375 μL of 10 mM AgNO3 aqueous solution was added into the reflux solution, which was stirred for 30 min to get a uniform mixture of gold and silver ions. After that, 2.5 mL of
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1 wt % trisodium citrate aqueous solution was quickly added into the mixture, and the color of the solution changed to orange gradually. The solution was refluxed for another 30 min and then was cooled to room temperature. UVvis spectrometer (PerkinElmer Lambda 35) was used to study the absorption of Au0.7Ag0.3 NPs in solution. Transmission electron microscope (TEM, Jeol 2100) was used to characterize the Au0.7Ag0.3 NPs. Surface Modification of SiO2 Substrates. A piece of 1 1 cm2 Si wafer with 300 nm thermally oxidized SiO2 was cleaned in a mixture of water, 30% H2O2, and 25% ammonia solution (5:1:1 v:v:v) at 80 °C for 5 min. Then, it was rinsed copiously with Milli-Q water three times. After the cleaned substrate was dried by N2, it was immersed into 42.5 mM 3-aminopropyltriethoxysilane (APTES) aqueous solution for 5 min to form APTES self-assembled monolayers (SAMs).25,46 The SiO2/AuAg NP substrate was obtained by immersing the APTES-modified SiO2 into Au0.7Ag0.3 NP suspension for 10 min and was rinsed with Milli-Q water. Fabrication of SiO2/FGO/AuAg NP Substrates. The APTES-modified SiO2 substrates were immersed into the activated GO suspension for 10 min. A drop of aqueous mixture of 0.05 M N-hydroxysuccinimide (NHS) and 0.2 M N,N0 -dicyclohexylcarbodiimide (EDC) was introduced onto the GO surface and was kept for 20 min to form reactive succinimide esters. Then, the substrate was thoroughly rinsed with Milli-Q water and was covered with 100 μL 99.9% ethylenediamine (NH2CH2 CH2NH2) for 30 min.30 One amine group of ethylene diamine reacted with the reactive succinimide ester on GO surface to form the SiO2/FGO substrate. The other amine group on SiO2/FGO substrate was used to attract negatively charged Au0.7Ag0.3 NPs later. After the substrate was rinsed with Milli-Q water, it was incubated in 0.5 mL as-prepared Au0.7Ag0.3 NP suspension for 10 min, which was then rinsed with Milli-Q water and was dried with N2 to obtain the SiO2/FGO/AuAg NP substrate. Characterization of Substrates Modified with Dye. A drop of sodium bicarbonate buffer solution (∼500 μL) containing 1 μM Alexa fluor@488 carboxylic acid, succinimidyl ester (abbreviated as Alexa fluor 488 in the main text) was dropped onto the APTES-modified SiO2, SiO2/AuAg NP, SiO2/FGO, and SiO2/FGO/AuAg NP substrates. After 10 min, the substrates were carefully rinsed with Milli-Q water. Field emission scanning electron microscope (FESEM, Jeol 7600F) was used to characterize GO adsorbed on APTESmodified SiO2 substrate, SiO2/AuAg NP substrate, and SiO2/FGO/AuAg NP substrate. Five kilovolts was used for imaging, and 20 kV was used for EDX element analysis. Raman spectra of Alexa fluor 488 on different substrates were taken on the WITec alpha 300 confocal Raman spectrometer. LASOS Argon-Ion Laser Power Supplies LGN 7812 with 488 nm was used as the power source. Low laser power (
