Enhancement in SERS intensity with hierarchical nanostructures by bimetallic deposition approach

Research Article

Received: 11 June 2011 Revised: 12 October 2011 Accepted: 7 November 2011 Published online in Wiley Online Library: 22 December 2011

(wileyonlinelibrary.com) DOI 10.1002/jrs.3128

Enhancement in SERS intensity withhierarchical nanostructures by bimetallicdeposition approachChit Yaw Fu,a,b† Kiang Wei Kho,b,c† U. S. Dinish,a Zhen Yu Kohe andOlivo Malinia,b,c,d*

We investigate the plasmonic enhancement arising from bimetallic (Au/Ag) hierarchical structure and address the fundamentalissues relating to the design of multilayered nanostructures for surface-enhanced Raman scattering (SERS) spectroscopy. SERS-
active nanosphere arrays with Ag underlayer and Au overlayer were systematically constructed, with the thickness of each layeraltered from 40 to 320nm. The SERS responses of the resultant bimetallic structures were measured with 2-naphthalenethiol dyeas the test sample. The results confirm the dependency of SERS enhancement on the thickness ratio (Au : Ag). Compared withAu-arrays, our optimized bimetallic structures, which exhibit nanoprotrusions on the nanospheres, were found to be 2.5 timesmore SERS enhancing, approaching the enhancement factor of an Ag-array. The elevated SERS is attributed to the formation ofeffective hot-spots associated with increased roughness of the outer Au film, resulting from subsequent sputtering of Au granuleson a roughened Ag surface. The morphology and reflectance studies suggest that the SERS hot-spots are distributed at thejunctions of interconnected nanospheres and over the nanosphere surface, depending on the thickness ratio between the Auand Ag layers. We show that, by varying the thickness ratio, it is possible to optimize the SERS enhancement factor withoutsignificantly altering the operating plasmon resonance wavelength, which is dictated solely by the size of the underlying nano-spheres template. In addition, our bimetallic substrates show long-term stability compared with previously reported Ag-arrays,whose SERS efficiency drops by 60% within a week because of oxidation. These findings demonstrate the potential of using sucha bimetallic configuration to morphologically optimize any SERS substrate for sensing applications that demand huge SERSenhancement and adequate chemical stability. Copyright © 2011 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.

Keywords: bimetallic substrate; metal film; surface-enhanced Raman scattering; stability; hierarchical structures; biosensing

* Correspondence to: Olivo Malini, School of Physics, National University of Ireland,Galway, Ireland. E-mail: [email protected]

† These authors contributed equally to this work.

a Bio-optical Imaging Group, Singapore Bioimaging Consortium, Agency forScience Technology and Research, 11 Biopolis Way, Singapore, 138667

b School of Physics, National University of Ireland, Galway, Ireland

c National Cancer Centre Singapore, 11 Hospital drive, Singapore, 169610

d Department of Pharmacy, National University of Singapore, 18 Science Drive 4,Singapore, 117543

e Division of Bioengineering, Block E3A, #04-15, 7 Engineering Drive 1, NationalUniversity of Singapore, Singapore, 117574

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Introduction

By a phenomenon known as surface-enhanced Raman scattering(SERS), nanoroughened substrates offer giant field enhancementfor high sensitivity detection of weak Raman scattering.[1] Sincethe discovery of SERS, the use of corrugated noble-metal surfacesor nanoparticles as a biosensing tool[2,3] has gained popularity. Inaddition to molecular fingerprinting with SERS, the narrow spectralwidth permits multiplexed detection.[4]

Most SERS substrates are composed of metallic colloid[5] or pat-terned solid surfaces.[6] In particular, periodic nanostructures capableof sustaining reproducible field confinements and highly intensefields are necessary in SERS. Gold (Au) and silver (Ag) are preferredas SERS-supporting materials because of their favorable opticalcharacteristic.[1] Although gold colloids have been used to targetanimal tumors,[4,7] a warrant control of colloidal stability underin vivo conditions remains a challenge.[8] On the contrary, patternedsolid substrates such as sphere-templated films,[9] nanogap-structured films,[10] biomimetic substrates,[11] porous films,[12] andnanoparticle-anchored surfaces[13,14] are immune to aggregationand are thus ideal for ex vivo biosensing.[3] Among these substrates,metal film over nanosphere (MFON) has been amply demonstratedto exhibit reproducible and predictable Raman enhancement.[3]

MFON has been devised by Van Duyne using an effective fabricationprocess inwhich polystyrene (PS) nanospheres are self-assembled on

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a planar solid support and then followed by metal coating.[9] Thiswell-defined surface patterning approach is economical and lesslaborious because the need for costly electron beam lithographyand intensive chemical treatments are omitted. These facts have thusmotivated many attempts to achieve prudent manipulation in PScolloidal arrangement.[15–17]

Apart from periodic structures, the coating thickness and compo-sition of the substrate surfaces are also important in optimizingSERS.[18–20] Recently, multilayered MFON has been shown to

Copyright © 2011 John Wiley & Sons, Ltd.

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improve its SERS enhancement in comparison to a single-layeredsubstrate though the factors that govern the additional enhance-ment are yet fully understood.[21] For instance, Cullum et al.developed MFON with 1-nm-thick silver oxide layers sandwichedbetween multiple layers of gold film.[22] Interestingly, it was foundthat the oxide spacers could promote interfacial electromagnetic(EM) couplings between neighboring gold films, which results in aSERS enhancement significantly higher compared with a single-layered MFON. Masson et al., on the other hand, studied a SERSsystem with alternating Ag and Au layers with each layer thicknessin the range of a few tens of nanometers.[19] It was shown thatincreased SERS activities can be attained from a bilayered substratewith 175-nm-thick bimetallic layer comprised of a Ag underlayerand a Au overlayer. Despite the structural similarity in these twoworks, it appears that different stratified configurations can leadto fundamentally distinct optical properties mainly because of thelimited skin depth and Plasmon hybridization.[23] To fully harnessthe benefits from those configurations, it is thus necessary to con-duct an in-depth study to understand the underlying mechanismof the additional SERS resulting from the multilayered structures.In this paper, the fundamental architectural design of such

multilayered substrate is addressed by using the bimetallic MFON(BMFON) substrates as a study model. First, single-layered MFONsubstrates with either Ag or Au film were studied as a functionof metal thickness. These optimized substrates were comparedwith BMFON. Next, we investigated the SERS activity of BMFONsubstrates ‘decorated’ with nanoprotrusions (NPRs) derived byoverlaying a Au layer onto a Ag-coated nanosphere array. Theuse of an outer Au layer is advantageous to biosensing becauseof its binding affinity to thiol groups that assist in bioconjugation,and to its ability to protect the underlying Ag layer from oxidation,thereby extending the shelf-life of the substrate for long-termmonitoring applications. We showed that there exists an optimalAu : Ag thickness ratio where the BMFON substrate exhibits SERSenhancement and stability exceeding those of the Au-coatedMFON. We first optimize the SERS enhancement of the first Aglayer by tuning the surface morphology, i.e. the size of Ag-NPRon the PS spheres. These preformed Ag-NPRs were then used asa ‘morphological template’ for the subsequent Au deposition. Asthe amount of Au deposited increased, so did the size of theresultant Au–Ag bimetallic NPRs, which concomitantly resultedin narrowing of the gaps between the adjacent NPRs. Theresultant SERS enhancement can be attributed to surfacemorphological changes that, in turn, regulate the number of effec-tive hot-spots. The most significant observation is that this two-step morphological optimization only minimally shifts theplasmonic resonant wavelength, thereby allowing an indepen-dent plasmonic tuning solely by adjusting the size of thenanosphere. We conclude that the plasmonic properties of ourbimetallic substrate can be morphologically optimized for sensingapplications that demand huge SERS enhancement and adequatechemical stability.

Experimental section

Metal film over nanosphere substrate preparation

Monodisperse PS colloidal suspension (�=384nm, 2.5 wt%) waspurchased from Kisker and stored at 4 �C when not in use. Priorto use, the PS sphere solution was added with 15 wt% sodiumdodecyl sulfate (SDS), a surfactant, to yield a composite solutioncontaining 2.36 wt% PS spheres and 0.85 wt% SDS. Uncoated

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microscope glass slides were cut into square pieces (10mm� 10mm� 1mm) and used as the supporting substrate. The glass slideswere sonicated in a bath of ethanol for 5 min and dried with argongas. The PS sphere monolayers were prepared on these cleanedglass substrates by spin-coating method.[24] Next, 10 mL of theprepared colloidal solution was dispersed onto the center of a glassslide. A spin coater was used to spin coat each glass slide with thedroplet at 2000 rpm for 20 s. A green reflective surface can beimmediately observed because of the Bragg diffraction given bythe closely-packed PS spheres. The sphere-templated substrateswere then dried in a vacuum desiccator overnight. Finally, thesubstrates were coated with Au or/and Ag (99.999% purity, JEOL)at various thicknesses by sputtering technique (JEOL, JFC-1600Auto fine coater). Each metal layer was deposited at a rate of1.33 nm/s. The substrates of single metallic coating of Ag and Auare subsequently referred to as AgFON and AuFON substrates,respectively. On the other hand, substrates with bimetallic coatingare labeled as BMFON.

Morphological characterization

A scanning electron microscope (JEOL, SEM6340F) was used toexamine the surface morphology of the metallic substrates, partic-ularly at SERS-active regions where the PS beads were packed.Atomic force microscopy (AFM, Veeco Nanoscope IV MultimodeAFM) measurements were performed in tapping mode to investi-gate the surface roughness of the SERS substrates over an area of1mm.[2] A silicon AFM tip (RTESPA, Veeco, USA) with typicalresonant frequency of 300 kHz, force constant of 40N/m andcurvature radius of 8 nmwas used. The surface topologymaps wereprocessed and analyzed using image processing software (WSXMV5.0, Nanotec Electronica).

Raman spectral measurement

Raman spectral measurements were carried out using a Ramanmicroscope (Invia, Renishaw, UK) equipped with a grating (1200line/mm, spectral resolution at 1 cm–1) and a charge-coupled devicecooled at �70 �C. A HeNe laser emitting at 633nm (Renishaw, UK)was used to irradiate the active region of the substrates via anobjective (50�, numerical aperture 0.75, Leica). The focus spot sizewas estimated to be 1mm. The laser-induced Raman signal wascollected through the same objective and detected in a backscatter-ing geometry. Prior to measurements, the instrument was calibratedwith the dominant Raman peak of silicon at 520 cm–1. 2-Naphthale-nethiol (NT, Sigma-Aldrich) was used as the probe molecule and10mM stock solutions were prepared in ethanol. Each SERS substratewas incubated in NT solution at room temperature for 12 h.Following that, the substrate was rinsed copiously with ethanol toremove the unbound NT molecules and dried with argon gas. SERSspectrumwas acquired at an optical power of 60mWwith an integra-tion time of 10 s. The shutter of the laser was immediately closedafter each measurement to minimize any possible photodamage tothe samples under a prolonged illumination. Baseline correction ofthe measured spectra was performed to remove the broadfluorescence band for data analysis. To evaluate the reproducibilityof the measurement, spectra were measured from ten randomlocations that are at least 10mm apart on each substrate. The relativestandard deviation of the intensity value was then calculated accord-ingly. Throughout the data analysis, the intensity value of NT at1379cm–1 was selected to compare the SERS enhancement of thesubstrates. The same procedure was implemented to conduct the

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Enhancement in SERS intensity with hierarchical nanostructures

stability test. In this study, substrates were stored for a specificduration of time before NT treatment followed by the Ramanmeasurement. To quantify the SERS enhancement, the unenhancedRaman spectrum was measured from a droplet of 100mM NTpipetted on a glass slide.

Diffuse reflectance measurements

Reflectance measurements of SERS substrates were performedusing a portable spectrometer (USB4000, Ocean Optics) coupledto a tungsten halogen light source (HL-2000-FHSA, Ocean Optics)and a reflection probe (QR400-7-SR, Ocean Optics). The reflectionprobe consists of a single collection fiber surrounded by six illumi-nation fibers. Each fiber has a diameter has a core diameter of400mm. All reflectance spectra were referenced against a metal-coated glass slide with the same coating thickness as the respectiveSERS substrate.

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Results and discussion

Figure 1 shows the formation of large domains of hexagonally-packed two-dimensional colloidal crystals by self-assembly process.The surface features are complex, but can be viewed as a superpo-sition of two periodic arrays of corrugated hemispherical metallicshells and pyramidal structures.[9] The nanosphere arrays withincreasing Ag-overcoat thickness exhibit rich surfacemorphologies.The AgFON presents smooth surface roughness and the interbeadgaps can be clearly discerned as shown in Fig. 1(a). To obtain bime-tallic substrates, Au of various thicknesses was subsequentlysputtered over the AgFON. As the deposition time increased, theNPRs on BMFON surfaces became more pronounced and beganfilling up the interbead gap as evident in Figs 1(b)–(e). The gap sizewas found to reduce approximately from 50 to 10nm.

In Fig. 2, we compare the 1mm� 1mm AFM maps of AgFON andBMFON substrates, and their surface profiles as given in Fig. 3. Forma-tion of the NPRs on the bead can be clearly seen. Silver particles ofaround 50nm are present on the bead surface of AgFON. Enlargedbimetallic NPRs, with outer gold shell and silver core become evidentas the AgFON was gold-coated as illustrated in Figs 2(b) and (c). Theprogressive growth of bimetallic NPRs was also accompanied by areduction in their number, suggesting that these growing bimetallicNPRs coalesced into larger ones with a prolonged sputtering process.The diameters of the bimetallic particles increased from 60 to 85nmas the gold over-coating increased from 240 to 320nm as estimatedfrom the AFM images. A close inspection of the PS bead surfaces andthe crevices is given in Fig. 3. Prior to Au deposition, we notedthe presence of deep crevices between the beads on AgFON.On the contrary, noticeable NPRs were seen between thebeads on the BMFON, which resulted in shallower crevices.Shallow crevices were formed during prolonged sputteringwhen two approaching NPRs coalesced into a larger grain atthe crevice as depicted in Fig. 3(d). The result suggests thata controlled coating can facilitate the formation of hierarchicalsurface roughness as the optical-enhancing features for SERS.

Surface-enhanced Raman scattering enhancement is traditionallyevaluated by measuring the enhanced signal strengths fromsubstrates coated with an adsorbed layer of Raman dyes such ascrystal violet and rhodamine 6G.[25] Chemiabsorbed dyes with a thiolgroup (S–H) are generally preferred for this purpose, because theyform continuous self-assembledmonolayers. In this study, NT, a thio-lated compound, is chosen to assess the enhancement performance

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because its SERS characteristics are well established in theliterature.[26] Because the excitation wavelength (633nm) used inthe current experiment is spectrally far away from the 241-nm elec-tronic absorption band of NT,[27] resonance enhancement frommolecules can be neglected and the observed enhancement canbe attributed entirely to EM enhancement from the substrates. Arepresentative SERS spectrumof NT is shown in Fig. 4(a). Its dominantRaman intensity at 1379 cm–1, which corresponds to the ring stretch-ing vibration,[28] was used to grade the SERS performance among theBMFON substrates of different design parameters.

Prior to discussion on BMFON, the SERS characteristics of single-layered substrates, AgFON and AuFON, were first studied in responseto different metal thicknesses. Their SERS performance was thenused for a comparison with BMFON. As shown in Figs 4(b) and (c),the SERS intensities as measured at 1379 cm–1 from the NT-treatedAgFONs and AuFONs are plotted against themetal thickness rangingfrom 40 to 320nm. In Fig. 4(b), it can be seen that the SERS intensityof AgFON begins to rise sharply with increasing Ag thickness from 50to 120nm, reaching the maximum enhancement at an Ag thicknessof about 160nm with an intensity value of 1.2� 105 counts.However, the intensity level drops by as much as 42% as thethickness increases further beyond 160 to 320nm. In a similarfashion, the SERS response of AuFON was also measured for variousAu thicknesses. The initial increase in SERS intensity was observed,reaching a maximum value of 3� 104 counts at 120nm thickness.Further increases in the thickness of the Au layer leads to dampingof enhancement with a shoulder located at a thickness of 280nm.It is noteworthy that the SERS intensities from AgFON is generallyabout 3–5 times those fromAuFON,which is in good agreementwiththe reported value.[29] Compared with AgFON substrate, enhance-ment observed from AuFON drops more abruptly as the Au layerincreases beyond the optimal thickness. Variation of intensity valueswas observed, but was, on average, within an interval of �15%.

Theoretical studies have shown that periodic assemblies of metallicparticles[30] and nanoshells[31] promote SERS, owing to the tightly-confined electromagnetic fields occurring at the crevices betweenparticles, particularly at those with acute angle intersection. Likewise,localized electromagnetic field or hot-spots are primarily distributedat the crevices between the PS beads. Recently, direct visualizationsof such intense hot-spots via Raman mapping were reported byFarcau and Astilean.[32] However, the impact of the metal thicknesson the hot-spot formation has not been extensively studied andaddressed. For instance, thin Ag coating thickness between 15 and25nm on AgFON was shown to give plasmonic peak matching withthe laser wavelengths at 633 and 785nm for increased SERS.[33]

Unfortunately, thin Ag coating can also give rise to the occurrenceof metal islands and pinholes, which results in the detection ofbackground Raman signals from the underlying PS beads. Anotherdisadvantage of a thin metallic layer, e.g. at thickness of 40nm, isthe small number of plasmonic hot-spots, which leads to weak SERSenhancement. On the other hand, we also show that excessivecoating of Ag or Au can weaken the SERS intensity (see Fig. 4). Thus,despite the ease of fabricating the nanosphere array, strongSERS enhancement is not guaranteed with arbitrary metalthickness. We postulate that the overall SERS performance iscritically related to the grain size of the material sputteredon the nanosphere surface.[34–36] With continuous sputtering,the discrete metal islands can eventually coalesce into alarger grain, and lose its nanometric features favorable forSERS. The SERS response profiles in Fig. 4 suggest theeffective use of metallic thickness to modulate the surfacetopology for optimal SERS.

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Figure 1. SEM micrographs of (a) AgFON substrate with 160-nm-thick Ag layer and (b)–(e) BMFON substrates with underlying Ag layer (160 nm) andupper Au layer at a thickness of (b) 40 nm, (c) 120 nm, (d) 240 nm, and (e) 320 nm. The inset in (a) is the enlarged figure of interbead gaps of nearly50 nm. A subsequent Au coating gradually reduces the gap size down to around 10 nm.

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Despite the fact that silver is an excellent Raman enhancing mate-rial, silver substrate works best only under a dry and oxygen-deficientenvironment.[37] The silver layer is prone to oxidation and becomesunstable in biological solutions such as phosphate buffer solution.[8]

The unfavorable condition can shorten the shelf-life and reduce SERSactivity on silver film over a period of time. The presence of silveroxide layer can further complicate the spectral analysis when thesubstrate is given prolonged optical exposure.[38] To this end, weinvestigate bimetallic coating in an attempt tomerge the advantagesfrom silver and gold. In particular, gold film was deposited over thesilver surface to protect it against oxidation. Compared with AuFONsubstrate, we demonstrate that the BMFON substrate offers a stableplatform for targeted sensing and bimetallic hierarchical structuresfor surpassing SERS enhancement.Two experiments were conducted to study the SERS response of

the individual layer of the bimetallic substrates by keeping one ofthe two metallic layers at optimal thickness and varying the other.Figure 5(a) depicts the intensity variation of BMFON substrate withvarying thickness of gold layer while the underlying Ag thicknesswas fixed at 160nm. The SERS intensity from BMFON is halved fromthe maximum of AgFON when only 40-nm-thick gold layer wascoated on silver layer. By contrast, this SERS intensity value is found


to be nearly 22 times higher than AuFON with the same Au thick-ness. We postulate that the Ag layer underneath is not fully coveredby the 40-nm-thick Au layer. Hence, the SERS enhancement couldbe primarily contributed by the exposed underlying Ag layer.

Further increasing of the top Au layer’s thickness actually reducedSERS drastically, and a considerable loss in SERS enhancementresulted as the Au layer increased beyond a critical thickness of200nm. Nevertheless, it is noteworthy that the enhancement ofour BMFONs at an Au thickness of 120nm exceeds that of theoptimized AuFON with the same Au thickness. The result suggeststhe influence of the underlying Ag layer on the SERS enhancementof the upper Au layer.

The results shown in Figs 1 and 2 are compelling evidence thatenlarged NPRs are formed within the crevices between PS beads. Itis thus probable that those discrete bimetallic NPRs are contributingto the overall enhancement. Similar to Ag-coated or Au-coated PSbead array, intense electromagnetic field can be localized withinthe crevices or gaps between the NPRs,[30] giving rise to increasedSERS. However, as the optimal thickness of bimetallic layer is reached,substantial hot-spots are constituted on the beads at the expense ofshallower crevices. Further growth of the gold overlayer eventuallyweakens the electromagnetic couplings between the Au–Ag layer


Figure 2. AFM maps of (a) AgFON substrate with 160-nm-thick Ag layer and (b,c) BMFON substrates with underlying Ag layer (160 nm) and upper Aulayer at a thickness of (b) 120 nm and (c) 320 nm, respectively. The height (nm) of the surface topology is displayed in different shades as indicated bythe shading bars. The grey lines denote the line profiles used for morphological comparison in the next figure.

Figure 3. (a)–(c) Height profiles of the respective substrates as denoted by the grey lines in subfigures of Fig. 2. The crevice angle is illustrated by theangle between the two dashed lines in (a). The solid grey arrows indicate the NPRs formed at the surfaces and the crevices of PS beads. (d) Schematicrepresentation of the metallic shell around the three adjacent PS beads, demonstrating that the shell is not a perfect hemisphere. At increasing thick-ness, the smaller NPRs on the beads were enlarged with decreasing interparticle gap. The two approaching NPRs eventually coalesced into larger ones,yielding shallower crevices.


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Figure 4. (a) A representative baseline-corrected SERS spectrum of NT molecules measured from the BMFON substrates. The dominant Raman peak at1379 cm–1 is denoted in the circled region. SERS response curves at 1379 cm–1 of (b) AgFON and (c) AuFON substrates were plotted as a function ofmetal thickness ranging from 40 to 320 nm.

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and hence abolishes the optical properties of bimetal in accordancewith plasmon hybridization.[39] Additionally, the neighboringbimetallic NPRs on the beads also fused concomitantly to form largedeformed particles that reduce SERS.In the second study, BMFON was characterized by varying only

the thickness of the Ag layer while fixing the top Au layer thicknessat 120 nm (see Fig. 5(b)). This allows one to probe the effects of theunderlying Ag layer on the overall SERS enhancement. To this end,the outer Au thickness of bimetallic NPRs was kept constant, butthe size of inner Ag-NPRs was varied by sputtering process. The firstsilver sputtering provided a controlled number and size ofpreformed Ag-NPRs on the PS beads (see Fig. 3(a)). Gold films weresubsequently grafted on the Ag-NPRs during the secondary goldsputtering. Surprisingly, the enhancement increases by 67% when40-nm-thick Ag layer is predeposited beneath the 120-nm-thickAu layer , suggesting that the combined sputtering can be usedto engineer the surface topology favorable to SERS. In Fig. 5(b), atthe optimal condition where the Au : Ag thicknesses ratio is80 nm : 120nm, we note that the SERS enhancement level of theBMFON is between that of AgFON and AuFON. The enhancementratio of AgFON : BMFON : AuFON is calculated to be 1 : 0.60 : 0.25.This observation substantiates the feasibility of achieving bimetallicsubstrates with SERS enhancement close to that of silver-coatedsubstrates while using the over-coating Au film as a platform forbioconjugation. In our case, we believe plasmon hybridization ofthe bimetallic layer is not a contributory factor of the additionalSERS enhancement observed from BMFON. This is becauseplasmon hybridization typically arises when the metal thickness isbelow 10nm,[40] which is considerably lower than the thicknessused in our studies. It has been shown that roughness of goldnanolayer is highly dependent on the underlying surface.[41] Thus,the elevated SERS from BMFON can be attributed to the formation


of effective hot-spots associated with increased roughness of theouter Au layer (Figs 1 and 2), which resulted from sputtering ofthe Au layer over the roughened surface of the Ag. Our result couldbe used to substantiate the additional SERS intensities observedfrom multilayered Au–Ag substrates.[19,22] Masson et al. have alsoinvestigated the similar hierarchical substrates, but observed adrastic drop in their SERS enhancement factor (EF) compared withAuFON.[19] In particular, the SERS EF of BMFON is only about 30%that of AuFON. On the hand, we show that it is possible for BMFONto surpass the EF of optimized AuFON by choosing the appropriatebimetal thickness. Similar observation of optimal morphologyobserved fromAu–Ag colloid was reported and its SERS enhancementwas found to exceed that of Ag colloid.[42] Accordingly, simulation andfurther investigation of BMFON are in the pipeline to determine thepossible configuration to achieve SERS enhancement even greaterthan Ag substrate. While the bimetallic coating forms effective hot-spots, the PS spheres underneath are used to determine the operatingplasmonic wavelength.[29,43]

On the basis of the results in Figs 4 and 5, SERS EFs of the opti-mized AgFON (160nm), AuFON (120nm), and BMFON (Ag : Au=80nm : 120nm) are calculated to gauge their SERS performance. Thespatially-averaged EF, G, is determined by using the establishedmethod[44] of comparing the NT SERS intensity with a thin liquidlayer of NT. G can be expressed as

G ¼ISERSNSERS

� �IRamanNBulk

� � (1)

where IRaman and ISERS are the intensity values at the scattering bandof interest, i.e. at 1379 cm–1, in bulk liquid Raman spectrum andSERS spectrum respectively. NBulk is the number of NT molecules


Figure 5. SERS response curve of BMFON substrates as a function of (a) superficial Au layer thickness and (b) underlying Ag layer thickness. The Aglayer thickness was fixed at 160 nm in (a) and the Au layer thickness was set at 120 nm in (b).

Figure 6. (a) Normalized reflectance spectra (probability density func-tion) of BMFON with fixed thickness of Au layer at 120 nm and varyingthickness of Ag layer from 40 to 320 nm. The dotted lines denote the ex-citation wavelength, lex, and emission wavelength, lSERS. (b) Normalizedreflectance ( ) at wavelength of 633 nm plotted as a function of Aglayer thickness. The reflectance profile is well correlated with the SERS en-hancement trend ( ) that has been mentioned in Fig. 5(b).


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in bulk solution contributing to the unenhanced Raman signal,while NSERS is the number of NT molecules that are chemisorbedon the substrate and contributes to SERS signal. NRaman and NBulk

can be determined from experiments and G is rewritten as

G ¼ HrRm

� �ISERSIRaman

� �(2)

where R is the area factor and m is the packing density of NT mole-cules on the surface of the substrate.[27] H is the apparent height ofthe NT liquid layer emanating Raman signal and r is the moleculardensity of the NT solution prepared. The detailed calculation is givenin the supporting inormation. Studies elsewhere corroborated theexistence of hot-spots at the rim of PS.[32] We use a geometric modelfor the SERS-active surface from which R is estimated to be 0.05.Accordingly, G values of AgFON, AuFON, and BMFON are calculatedto be 3.5� 107, 9.0� 106, and 2� 107, respectively. Without theresonance enhancement from NT, these values primarily manifestthe substrate performance.

In this study, the AFM can hardly resolve the NPRs, particularly atthe crevices. The localized surface plasmon resonance (LSPR) relatingto optimal SERS can be associated with the minimum reflectivity ofSERS substrates.[3] Thus, reflectivity of BMFON was measured to cor-relate the SERS response curve with the resultant LSPR (Fig. 6). Thereflectivity dip shifts from 580 to 590nm as the thickness of Ag layerincreases from 40 to 120nm and the Au thickness is fixed at 120nm.However, further increase in Ag thickness reverses the dip spectralposition back to a shorter wavelength of 570nm. The initial red-shifted reflectance dip suggests that the NPRs that reside at thecrevices come closer.[45] The subsequent reverse of the dip positionindicates that the approaching NPRs eventually coalesce into a largerparticle which, in turn, forms shallower crevices and degrades thehot-spot in favor of SERS. While not presented here, the size of thePS bead can be selected to exhibit plasmonic peak matching withthe excitation wavelength used.[29] Li et al.[46] and Cintra et al.[47] havedemonstrated thatMFON substrates with diameter between 350 and420nm yield the greatest enhancement at excitation wavelength of633nm.

The normalized reflectance at the excitation wavelength is alsoplotted as a function of Ag thickness, as shown in Fig. 6(b). Notably,the absorbance resembles the SERS response of the substrates asgiven in Fig. 5(b). It is known that LSPR is sensitive to surface morphol-ogy and material compositions. Significant modulation in reflectancewith metal thickness were reported.[29,47,48] It is noteworthy that themeasured shift in LSPR of BMFON studied here is not significant,suggesting that the controlled dual sputtering can increase thenumber of strong hot-spots for greater SERS while retaining the


operating resonant frequency regulated by the PS beads. The negligi-ble shift in plasmonic peakwith increasing roughness is also consistentwith simulation studies of roughened nanoshells.[49] Thus, it is possiblefor one to tune the SERS enhancement factor of the BMFON, indepen-dent of its LSPR resonant frequency. This is in fact a unique feature ofour BMFON configuration. Nonetheless, we must stress that, althoughmatching the LSPR dip to the laser excitationwavelength can optimizeSERS activities, it is the total number of available plasmonic hot-spotsthat eventually dictates the final overall enhancement.[47] The total


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number of available hotspot is also a critical factor in deciding the finalSERS intensity. For instance, it is possible to obtain adequate enhance-ment from the substrate with more hotspots even while operatingoutside the optimal resonance condition.In addition to SERS enhancement, structural reproducibility[10,50]

and stability[22,46] of the substrate are also crucial in realizing a suc-cessful SERS-based sensing.[51] Although the use of the atomic layerof alumina on AgFON to preserve its SERS activity was reported,[52]

this method needs a precise control of the alumina thicknessbecause the SERS can be quenched exponentially with increasingthickness.[53] We have compared the shelf-life of optimized BMFON(80nm-Ag and 120nm-Au) and AgFON (160nm-Ag) as given inFig. S2. To achieve this, we first exposed these substrates to ambientenvironment for a period of time. The substrates were then treatedwith NT and the SERS intensity was measured to grade their stability.There was no significant degradation observed from BMFON and itsstability profile was fairly consistent with the reported findings on thestability of AuFON.[46] On the contrary, AgFON degraded rapidly,showing a reduction in SERS intensities to nearly 60% within a week.Similar observation of short shelf-life from AgFON was alsoreported.[46] Notably, the result substantiates the potential of BMFONfor use in long-term sensing. Thus, the increased stability is likely toallow extended chemical treatment for functionalizing the surface,particularly useful in biosensing applications. Most importantly,thiolated cancer markers, which readily attach to the gold surface,are available and extensively used in biosensors.[7,54]

Conclusion

We have developed BMFON substrates with bimetallic hierarchicalstructures exhibiting increased stability and optical enhancementcompared with substrates with a monolayer of Ag/Au coating.The BMFON substrates were fabricated using consecutive Ag andAu sputtering process from which Ag NPRs were preformed onthe densely-assembled nanospheres, followed by a surfaceshielding with Au layer. The SERS enhancement of conventionalAg-coated or Au-coated nanosphere-array was first studied atvarying thicknesses ranging from 40 to 320nm. The results showthat the enhancement of optimized Au-array is only at most aquarter of that of Ag-array, indicating the high dependency of SERSproperties on coating materials and thickness. Subsequently, asimple two-step sputtering approach has been demonstrated todevelop bimetallic NPRs on the surface of BMFON. The SERSenhancement of BMFON was then optimized via a progressivesputtering-growth of underlying Ag-NPRs and outer Au shells.Variations in SERS intensities were observed and correlated to theevidential changes in surface morphologies resulting fromsputtering growth. The results suggest that an optimal sputteringcondition could populate the amount of efficient hot-spots on thebimetallic surface, resulting in greater SERS enhancement.Compared with Au-array, optimized BMFON (80 nm-Ag and120nm-Au) yielded surpassing SERS intensity by 2.5 times andthe corresponding enhancement factor was estimated to be2� 107. We also showed that the stability of BMFON substrateswas comparable to the reported Au array whereas the SERSenhancement of Ag array was found to degrade within a week. Inaddition, we noted that the proposed fabrication scheme couldallow independent optimization of SERS enhancement andoperating resonance frequency. The results presented in this paperhas substantiated that the plasmonic properties of BMFON can begeometrically tuned for sensing applications that demand huge


SERS enhancement and adequate chemical stability. The proposedbimetallic deposition could be adapted for different periodicstructures to achieve the optimal performance.

Acknowledgements

We thank Quan Lam Zhung (Nanyang Technological University,Singapore), Dr Patricia Thong Soo-Ping (National Cancer Center,Singapore) and our groupmembers including Dr Praveen Thoniyot,Douglas Goh Wenda, Jason Soh Kiat-Seng and Shashi Rautela fortheir support in this work. Lastly, we acknowledge Tan Suat Hoonand Lu Thong Beng, from National University of Singapore, for theirtechnical assistance in surface characterization.

Supporting information

Supporting information may be found in the online version ofthis article.

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Enhancement in SERS intensity with hierarchical nanostructures by bimetallic deposition approach