This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Elaboration of an Ag°/TiO2 platform for DNA detection by surface enhancedRaman spectroscopy

Michel Langlet a, Idrissa Sow a, Samir Briche a, Mouna Messaoud a, Odette Chaix-Pluchery a,Francine Dherbey-Roussel b, Patrick Chaudouët a, Valérie Stambouli a,⁎a Laboratoire des Matériaux et du Génie Physique, Grenoble INP, CNRS, 3 parvis Louis Néel, 38016 Grenoble, Franceb Consortium des Moyens Technologiques Communs, 1260 Rue de la Piscine, Domaine Universitaire, 38402 Saint Martin D'Hères, France

a b s t r a c ta r t i c l e i n f o

Article history:Received 14 February 2011Accepted 16 August 2011Available online 24 August 2011

Keywords:SERSLabel free DNA detectionTiO2

Ag nanoparticlesSol–gel

The promising potential of a new platform for the label-free DNA detection by Surface Enhanced RamanSpectroscopy (SERS) is reported. This platform is composed of silver nanoparticles distributed over the sur-face of a TiO2 (anatase) film. DNA molecules are immobilized on the platform using a simple grafting pro-cedure. The elaboration and biomodification of the platform involve wet chemistry routes and benefitfrom specific properties of its components. First, the photocatalytic activity of TiO2 films is used for the pho-toreduction of Ag salts leading to the Ag° nanoparticle deposition. Second, the grafting and surface distribu-tion of DNA benefit from both the photo-induced hydrophilicity of TiO2 films and the chemical affinitybetween metallic silver and amine ligands. Despite a broad size distribution of the Ag° nanoparticleswhich affects the reproducibility of DNA Raman spectra, specific changes of DNA Raman line intensities ob-served in single strand and hybridized DNA attest that hybridization can reliably be detected and that a spa-tially addressed DNA detection is possible.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

In the field of biosensors, the label-free strategies to detect bio-molecules, and notably DNA hybridization, are under intense devel-opment. These strategies rely on the modification of a physicalparameter of the transducer in response to the biological event,namely changes in the refractive index in the case of Surface PlasmonResonance (SPR) [1–3], in the conductivity in the case of electrical orelectrochemical detection [4–6], in the resonance frequency in thecase of bio-MEMS [7,8]. In these techniques, the detected signal isnot necessarily specific of the molecule to be analyzed. In contrast,and among other advantages, the Surface Enhanced Raman Spectros-copy (SERS) method provides a powerful, rapid, and sensitive way toanalyze the structural fingerprint of molecules through the specificdetection of Raman peaks [9]. SERS is often used to detect DNA hy-bridization in colloidal solutions [10–13]. Studies also report DNA de-tection on substrates by SERS, but most of them mention the use of aRaman active label linked to DNA because it is more easily detectedthan the weak DNA Raman signal [14–16]. In this context, the devel-opment of functional and simple SERS platforms for the label-free

DNA detection remains a challenge of prime interest. Different strat-egies have been proposed: some authors elaborate silver mirror sub-strates [17] or electrochemically roughened silver sheets [18]. Othersreport a very high SERS activity of optimized SiO2/Si substrates coatedwith Ag° nanoparticles (NPs) [9] elaborated with a rather complexand expensive multi-step vacuum procedure [19]. More recently,the decoration of vertically aligned Si nanowires (SiNWs) with AgNPs has been used to generate unique SERS substrates for bio-analytical measurements [20,21]. However, the controlled elabora-tion of NWs requires a specific know-how. Other papers report nearlycontinuous Ag coatings [22] or Ag clusters [23] deposited on TiO2 orMgO films by using sol–gel and photo-reduction processes. Suchfilms exhibit a high SERS activity to detect organic molecules butthey have not been tested for DNA detection.

Alternatively, we propose a new platform made of Ag° NPs dis-persed on a TiO2 film surface in view of a label-free SERS detectionof DNA. Our work mainly focuses on the elaboration of the platformand its biomodification by DNA. It reports the first reliable Ramanspectra of DNA grafted on this platform. Assets of this concept com-prise the implementation of low cost, simple, and flexible wet chem-istry routes, in association with specific photo-induced propertiesarising from the TiO2 (anatase) films. In a previous study, we havedemonstrated that these films deposited using the sol–gel techniqueexhibit excellent photo-induced characteristics such as the photoca-talytic activity and the photo-hydrophilicity [24]. These propertiescome from the generation of electron-hole charge carriers when the

Surface Science 605 (2011) 2067–2072

⁎ Corresponding author.E-mail address: valerie.stambouli-sene@grenoble-inp.fr (V. Stambouli).

0039-6028/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.susc.2011.08.007

Contents lists available at SciVerse ScienceDirect

Surface Science

j ourna l homepage: www.e lsev ie r .com/ locate /susc

Author's personal copy

films are exposed to UV light of energy greater than the band gap ofanatase (EgN3.2 eV; λb380 nm). In this paper, we report how theseproperties play a key role in both the elaboration and the biomodifi-cation of the platform.

2. Experimental

2.1. Sol formulation and TiO2 film processing

Crystalline TiO2 films of 70–80 nm thickness were firstly depositedthrough two sol–gel routes. The first route is based on a protocol de-tailed in a previous article (route I in Fig. 1) [24]. Briefly, a polymericsol (sol 1) was prepared by mixing tetraisopropyl orthotitanate (TIPT;Ti(C3H7O)4 from Fluka) with deionized water, hydrochloric acid, andabsolute ethanol as a solvent. The TIPT/H2O/HCl/ethanol molar compo-sition was 1/0.82/0.13/43. The sol 1 was then deposited at room tem-perature on (100) silicon wafers (3.3×3.3 cm2) by spin-coating usinga Suss Microtec RC8 apparatus. Prior to deposition, the substrateswere cleaned with ethanol, then rinsed with deionized water, anddried with air spray. For each deposition, 300 μl of sol were spread onthe substrate rotated at 3000 rpm. After liquid film deposition, the sol-vent rapidly evaporated and a homogeneous xerogelfilm formed at am-bient atmosphere through the well-known sol–gel polymerizationroute. The xerogel film was annealed at 500 °C and changed into afilm crystallized in the anatase polymorphic form of TiO2.

Alternatively, we tested a second sol–gel approach to form pat-terned TiO2 films (route II, Fig. 1). In that case, the previously de-scribed polymeric sol 1 was modified using a complexing ligand(Benzoylacetone (BzAc) from Aldrich). The whole protocol has beendetailed in a previous article [25]. Briefly, a sol was prepared usingTIPT, BzAc, and methanol (MeOH) as a solvent. The TIPT/BzAc/MeOHmolar composition in this sol was 1/0.91/20.4. The sol was thenmixed with the sol 1 to form a final sol 2 with a TIPT concentration

of 0.6 M and a BzAc/TIPT molar ratio of 0.6. The sol 2 was then spin-coated on silicon wafers in conditions similar to those used for thesol 1. Here again, this protocol yielded the deposition at room tem-perature of homogeneous xerogel films. However, complexation ofTIPT with BzAc strongly reduces the sol–gel reactivity of the titaniumprecursor. Owing to the poor chemical stability of the resulting Ti–BzAc complex, derived xerogel films deposited at room temperaturecan easily be leached through simple ethanol washing. Conversely,an exposure of such films to UVA light induces a photolytic decompo-sition of the Ti–BzAc complex, leading to an enhanced chemicalstability of the film toward ethanol washing. A selective UVA exposi-tion through a mask, associated to solubility contrasts betweenareas exposed or not to UV light, finally enabled us to implement aprocedure leading to a fine photo-patterning of sol–gel films througha simplified one-step protocol [25]. TiO2 patterned films can in turnbe crystallized in the anatase form through a heat-treatment at500 °C yielding photocatalytic and photo-hydrophilic TiO2 patterns.In this study, this protocol was used to pattern circular TiO2 spots of150 μm diameter.

2.2. Photo-generation of silver NPs

To disperse Ag° NPs on the surface of patterned or unpatternedTiO2 films (routes I and II in Fig. 1), we have taken advantage of thephotocatalytic activity of the TiO2 films which only occurs when thefilms are exposed to UVA wavelengths lower than 380 nm. A liquidsolution of AgNO3 diluted at 0.1 M in a 80/20 water/ethanol mixturewas exposed for 5 min to UVA light provided by PLS-11 W lamps(Philips) in the presence of a TiO2 film. These lamps present a contin-uous UVA emission spectrum, centered at around 365 nm, and theydo not emit in the UVB and UVC ranges. We have previously shownthat this procedure involves a photo-reduction mechanism ofAgNO3 adsorbed at the TiO2 film surface, promoted by electrons

TiO2 film

Si substrate

AgNPs

SiO O O NH2

NH

NH2NH2 NH2 NH2

Route II

Ia Ib

DNAprobes

Sol 1 Sol 2

500°

500°

AgNO3UVA

Route I

3, UVA

Ethanol leaching

maskUVA

Sol 1 Sol 2

AgNO

Fig. 1. Different pathways involved in the elaboration and biomodification of the Ag°/TiO2 platform. Route I: TiO2 film elaborated from sol 1, Ag° NPs deposition onto the TiO2 surfacefollowed either by a multi-step DNA probe immobilization procedure including silanization (route Ia), or by a direct DNA probe immobilization procedure (route Ib). Route II:photo-patterning of the TiO2 film elaborated from sol 2 followed by Ag° NPs deposition and subsequent biomodification using a direct DNA probe immobilization procedure.

2068 M. Langlet et al. / Surface Science 605 (2011) 2067–2072

Author's personal copy

photo-generated by the TiO2 film, and yielding metallic Ag° NPs dis-tributed over the TiO2 surface [26].

2.3. DNA immobilization

Ag°/TiO2 heterostructures were bio-modified with DNA molecules.A standard multi-step time consuming procedure used in the case ofSiO2 and metal oxide films [27] was first employed for grafting unpat-terned films (route Ia in Fig. 1). A silanization step performed throughthe deposition of 3-aminopropyl-triethoxysilane (APTES) in ethanolwas followed by successive rinses in order to remove the unbound si-lane and by curing at 110 °C. Then, the films were functionalized withsingle-strand (ss) DNA probes consisting of pre-synthesizedmolecules with 20 bases (5′ NH2-TTTTT-GATAAACCCACTCTA 3′)(Apibio/BioMérieux, France): the probes were diluted to aconcentration of 100 μM with sodium phosphate solution in water.20 μl drops of this solution were manually deposited on the surface ofeach sample before an overnight incubation at room temperature.Prior to this step, the DNA grafting on the silanized surface wasobtained by using a cross-linker molecule (glutaraldehyde) which ledto a strong covalent bond between the NH2 termination of APTES andthe 5′NH2 termination of the DNA probes. A second and considerablysimplified procedure consisting of the direct grafting of DNA onAg°/TiO2 heterostructure will be presented and discussed further on.

The double-strand (ds) DNA molecules were obtained after hybrid-ization of DNA probes with a complementary target sequence (5′ CAT-AGAGTGGGTTTATCCA 3′) (Eurogentec, Belgium) diluted to aconcentration of 10 μM in a phosphate buffer solution (pH 5.5). 20 μldrops of the target DNAwere deposited over the whole sample surface.The hybridization reactionwas performed at 42 °C for 45 min. The sam-ples were then rinsed with different saline solutions in order to removeunhybridized target DNA molecules: first, SSC (saline sodium citratebuffer solution 2× concentrate), second, SDS (sodium dodecyl sulfatesolution) and again SSC (0.2× concentrate). The samples were finallydried under Ar gas. For preliminary investigations, the target sequenceswere labeled with a fluorophore (Cy3) to follow efficient DNA graftingand hybridization by fluorescence imaging.

2.4. Characterization

Morphology and chemistry features of TiO2 films and Ag° NPs wereanalyzed by Field Emission Gun-Scanning Electron Microscopy (FEG-SEM) using a controlled pressure FEI Quanta FEG 250. The microscopeequippedwith an energy-dispersive X-ray (EDX) detectorwas operatedat 3 kV and 120 Pa in secondary electron mode. Circular spots photo-patterned on TiO2 filmswere imaged using a LeicaDMLMopticalmicro-scope. Water contact angle measurements (sessile droplet method;water pH 5.7) were performed at room temperature using a dropshape analysis system (Kruss DSA 10 MK2) connected with a videocamera. An average contact angle was determined from values mea-sured on the left and right sides of droplets of 0.5 μl deposited on differ-ent areas of the film surface. Fluorescence measurements wereperformed using an Olympus microscope (BXM41M), equipped with a100W mercury lamp, a cyanine Cy3 dichroïc cube filter (excitation550 nm, emission 580 nm), and a cooled Spot RT monochrome camera(Diagnostic). Raman measurements were carried out using a Jobin–Yvon/Horiba LabRam spectrometer equipped with a liquid nitrogenCCD detector. Experiments were conducted in micro-Raman mode atroom temperature. Spectrawere collected in a backscattering geometryusing the 632.8 nm line of a He–Ne laser. The light was focused to a1 μm2 spot using a 50× long working distance objective. The laserpower was 45 μW at the sample surface. Raman spectra have beenobtained for an acquisition time varying between 400 and 900 s. Theywere calibrated using the Si spectrum at room temperature.

3. Results and discussion

3.1. Surface characterization of the bare platform

The FEG-SEM image of Fig. 2a presents the typical morphology ofcrystalline TiO2 films (without Ag NPs) deposited from either sol 1or sol 2. The film exhibits a smooth surface with a fine and uniformgranular morphology. Fig. 2b shows the FEG-SEM image of silverNPs quite uniformly dispersed over the surface of a TiO2 film throughthe photocatalytic reduction mechanism. They exhibit a rather spher-ical shape with diameter comprised between a few tens to 100 nm ormore, which constitutes a rather broad size distribution. Some oblongparticles can also be punctually observed. Since this work is focusedon the feasibility study of DNA detection using our SERS platform,no particular attempt has yet been made to optimize the morphologyof Ag° NPs. A similar morphology is observed in the case of silver NPsdispersed over crystalline and patterned TiO2 films deposited fromthe sol 2. Grazing incidence X-ray patterns indicated that silver parti-cles dispersed over the TiO2 surface crystallized in the face-centered

100 nm

500 nm

150 µm

b

c

a

Fig. 2. FEG-SEM images of the TiO2 film surface before (a) and after (b) deposition ofAg° NPs (bright spots). Optical micrograph of photo-patterned Ag°/TiO2 spots (darkareas) with bare silicon substrate in the inter-plots regions (c).

2069M. Langlet et al. / Surface Science 605 (2011) 2067–2072

Author's personal copy

cubic structure of metallic silver (ICDD card # 04-001-2617; not illus-trated here), which confirms that the photocatalytic reduction effi-ciently promotes the formation of metallic Ag° NPs. The opticalmicrograph of Fig. 2c shows circular Ag°/TiO2 spots formed usingthe photo-patterning procedure (route II in Fig. 1). Inter-plot regionscorrespond to the bare silicon substrate. Energy dispersive X-ray an-alyses revealed that Ag° NPs are located only on the surface of pat-terned spots (not illustrated here), which indicates that no photo-reduction mechanism yielding the formation of such NPs takes placeat the surface of the bare silicon substrate.

3.2. DNA immobilization on the platform: preliminary studies

The DNA probe grafting was achieved using the standard multi-stepprocedure including a silanization step as described in Section 2.3. Thetypical Raman spectrum of a bare TiO2 film (without Ag°) after DNA hy-bridization with Cy3 labeled DNA is shown in Fig. 3. This spectrum ex-hibits Raman lines related only to the Si substrate (520 cm−1+threesecond-order lines) and to the anatase TiO2 film (142 and 638 cm−1).No Raman signal related to DNA or Cy3 is observed although a strongfluorescence arising from the Cy3 fluorophore proves the grafting andhybridization of DNA sequences on the TiO2 surface (see inset inFig. 3). In contrast, Raman spectra measured after DNA grafting and hy-bridization on Ag°/TiO2 heterostructures showed many lines assignedto Cy3 and DNA. This clearly indicates that the DNA and Cy3 detectioncan only be achieved by Raman spectroscopy in the presence of Ag°NPs giving rise to the SERS effect. However, spectra collected at differentpoints of the films differ in their line content and intensity emphasizinga lack of homogeneity. It could be explained by a heterogeneous DNAsurface distribution on the film surface. Indeed, it is known that a strongsurface tension aqueous medium traditionally forms a 3D drop on asolid surface, which is characterized by a given water contact angle. Inthe case of DNA probes on the Ag°/TiO2 surface, wemeasured a contactangle of around 50–60°. After water evaporation, the liquid drop led tothe spread of DNA in the formof a circular dot, as depicted in the inset ofFig. 3. Besides, it is also known thatwhen a colloidal suspension inwaterforms a 3D drop on a solid surface, the liquidmediumundergoes subse-quent radial evaporation, which involves competitive hydrodynamics,heat and mass transfer, and physico-chemical mechanisms [28]. Thesecompetitive mechanisms finally lead to a localized and non homoge-neous deposition of the dry matter on the solid surface. Thus, we inferthat such competitive mechanisms can explain the non uniform DNAgrafting at the Ag°/TiO2 surface.

3.3. DNA immobilization on the platform: new experimental approaches

To improve the DNA surface coverage homogeneity, a second andquite simple biomodification procedure was investigated in relation totwo additional specificities of the Ag°/TiO2 heterostructures. First, wetook advantage of the photo-induced property of TiO2 films, i.e. thephoto-induced hydrophilicity [24]. This property ensues from oxygenvacancies which are created at the TiO2 surface through oxydo-reduction mechanisms induced by electrons and holes photo-generated underUV exposure. Surface oxygen vacancies can then readilybe saturated by OH groups through a molecular or dissociative adsorp-tion of atmospheric water. It yields a super-hydrophilic surface, i.e. a sur-face with a strong affinity for water characterized by a water contactangle near zero degree. Since the photo-hydrophilicity is intrinsically in-duced by the TiO2 support, it points out the interest to preserve bare TiO2

areas by forming an island-like Ag° coating. In this new procedure, pat-terned or unpatterned Ag°/TiO2 heterostructures were then exposedfor 30 min to UVA light. A water contact angle of 4° was measuredafter UVA exposure, which shows the photo-induced super-hydrophilicity of our heterostructures. The second specificity of our sys-tem is based on the well-known chemical affinity of metallic silver foramine ligands, which promotes a bond linking of these ligands throughthe formation of Ag\N bonds [29]. Since amine ligandswere introducedat the 5′-end of (ss) DNA, we investigated the feasibility of a one-stepDNA grafting avoiding the previously described multi-step procedure.

DNAprobeswere grafted on the heterostructure surface immediate-ly after UVA exposure using this one-step procedure. In these new con-ditions, the DNA-containing aqueous drops naturally andinstantaneously spread at the surface of the superhydrophilic hetero-structures. It yielded a 2D liquid film which uniformly and totally cov-ered the sample surface, i.e. no circular boundaries seen in thefluorescence image of Fig. 3 could be observed after water evaporation.As a result, and contrary to the previous procedure, very similar DNAspectra could be acquired in different parts of the platform. It meansthat competitive mechanisms occurring in a 3D DNA liquid drop duringwater evaporation are probably reduced or canceled in the case of a 2Dliquid film, which leads to an improved oligonucleotide surface cover-age. This result proves i/the reliability of this simplified one-step graft-ing procedure, and ii/that the formation of a 2D liquid film efficientlypromotes the uniform immobilization of DNA probes.

Raman spectra of (ss) and (ds) Cy3-free DNA grafted using this one-step procedure are shown in Fig. 4a and b, respectively. In Fig. 4a, DNA isclearly identified by four main Raman modes. Three of them, at731 cm−1 and at 1310/1328 cm−1 (doublet) in the figure, are assigned

200

400

600

800

200 400 600 800 1000 1200 1400 1600

142

302

638

Ana

tase

Ana

tase

Si

SiSi

520

Si

Inte

nsity

Wavenumber (cm-1)

DNA

TiO2

DNA

TiO2

Fig. 3. Raman spectrum (counting time=400 s) and fluorescence microscopy image(inset) obtained after DNA probe grafting and hybridization with Cy3 labeled DNA tar-gets on a TiO2 film without Ag°. DNA immobilization was performed using a multi-stepprocedure. Lines are assigned to the Si substrate and TiO2 film Raman modes. In theinset, a part of the circular area coated with fluorescent DNA appears bright, whilethe uncoated TiO2 film appears dark (magnification ×200).

800 1000 1200 1400 1600

731

784

A C A

1310 13

28

(a)

(b)

(c)

SiInte

nsity

(ar

b. u

nits

)

1045

858

1096

1230

1450

1510

1580

1405

Wavenumber (cm-1)

Fig. 4. Raman spectra (counting time=900 s) of single strand (a) and label-free doublestrand (b) DNA grafted on an Ag°/TiO2 heterostructure. The DNA immobilization wasperformed using a single-step procedure. Main lines are assigned to Adenine and Cyto-sine Raman modes. (c) Raman spectrum of the Ag°/TiO2 heterostructure without DNAgrafting where only the second order Si Raman spectrum is visible.

2070 M. Langlet et al. / Surface Science 605 (2011) 2067–2072

Author's personal copy

to Adenine (A) as a breathing vibration mode and mixed in-planestretching motions of the ring skeleton, respectively [9]. The line at784 cm−1 is assigned to the breathing vibration mode of Cytosine (C)[9]. No Raman line related to Thymine and Guanine bases has beenclearly identified, probably due to their weak intensity. The dominanceof the A signal, in comparisonwith that of other DNAbases, probably re-sults from the higher SERS cross section of this base (ANCNNG≥T in anequimolarmixture of poly(A), poly(G), poly(C) and poly(T)) [9,30]. Theother lines observed in Fig. 4a and b are not easy to identify but all ofthem arise from DNA as attested by the spectrum of the Ag°/TiO2 het-erostructure submitted to the one-step procedure in the absence ofDNA, which only shows the high wavenumber line of the Si second-order Raman spectrum (Fig. 4c). The comparison between (ss) and(ds) DNA spectra indicates significant modifications in the relative in-tensities of A and C lines located near 731 cm−1 and 784 cm−1, respec-tively. Accordingly, the A:C intensity ratio appears to be about 2:1 in thecase of (ss) DNA probes (Fig. 4a), while it is closer to 1:1 after DNA hy-bridization (Fig. 4b). A:C intensity changes of Raman lines accompany-ing hybridization have also been reported by M. Green et al. for otherDNA sequences, the intensity ratio depending on the sequence [9]. Inour case, a quantitative validation of the intensity changes has beenobtained from three different spectra for each (ss) and (ds) DNA sampleafter baseline subtraction and linefitting. In the case of (ss)DNA, the av-erage A:C intensity (integrated intensity) ratio is found to be 1.9±0.1(0.8±0.1) whereas it decreases to 1.0±0.1 (0.4±0.1) for (ds) DNA.These intensity ratios are very close to the qualitative ratios estimatedfrom Fig. 4a and b. The conformational change induced by hybridizationresults in a rigidity increase of DNA strands and thus, in an increase ofthe distance between A bases and the Ag° NPs coated surface, whichmay explain the weaker A intensity in the (ds) DNA. These specificchanges of both Raman lines observed on several (ss) and (ds) DNA-grafted films attest that hybridization can reliably be detected throughDNA Raman spectra measured from our SERS platform.

When designing biosensors, an important challenge relies on multi-plexed analyses in order to rapidly and easily identify numerous bio-molecules of different natures on a single sample [9,31]. One way toreach this objective is to create micro-arrays enabling a precise localiza-tion of bio-molecules and their spatially addressed detection. To this aim,patterned TiO2 films obtained from sol 2 were coated with Ag° NPsthrough a photo-reduction procedure to form Ag°/TiO2 spots (route IIin Fig. 1). The patterned heterostructures were then photo-hydrophilized under UVA light and biomodified with DNA through theone-stepprocedure. Raman spectra of (ds) DNA collected on three differ-ent Ag°/TiO2 spots of patterned heterostructures are shown in Fig. 5.

They clearly indicate the presence of DNA on the spot surfaces despitesome differences in the DNA peak intensities while no DNA could bedetected on bare silicon regions. The three spectra exhibit a bettersignal-to-noise ratio in average compared to the (ds) DNA spectrumfrom Fig. 4b, and, similarly, the A:C intensity ratio is reproducibly closeto 1:1. Calculations from line fitting give an average intensity ratio of0.90±0.03 and an average integrated intensity ratio of 0.45±0.06.These values differ slightly from the previous ones obtained from (ds)DNA grafted on un-patterned TiO2/Ag films but the reproducibility is im-proved due to an increased signal-to-noise ratio. These latter observa-tions i/depict a spot to spot reproducible detection of DNAhybridization, which in turn illustrates a rather homogeneous DNA sur-face distribution using our simplified one-step procedure, and ii/provethe feasibility of a reliable SERS detection on Ag°/TiO2 micro-arrayswith spatially addressed detection of DNA molecules.

Based on the assumption that, in the case of our one-step graftingprocedure, DNA is mainly bonded to Ag° NPs, the broad size distribu-tion of the Ag° NPs observed on the TiO2 surface (Fig. 2b) can affectthe reproducibility of DNA Raman spectra. On the one hand, the sizeof Ag NPs as well as their distribution on the TiO2 surface governsthe amount of sites available for DNA grafting and hence the amountof DNA to be detected by Raman spectroscopy. On the other hand,their morphology is also expected to intrinsically influence theirSERS activity [32–34]. These results stress the importance to performfurther studies in order to better control the morphology of Ag° NPs(shape, mean size, shape and size uniformity) and their distributionon the SERS platform. The influence of these parameters on theSERS activity and the detection performances of the platform (repro-ducibility, durability, sensitivity, selectivity) have to be investigatedand analyzed.

4. Conclusion

A feasibility study of DNA detection using a new SERS platform basedon the elaboration of Ag°/TiO2 heterostructures through wet chemistryroutes is reported. It benefits from specific properties of the platformcomponents, such as the photo-induced properties of TiO2 films andthe chemical affinity between silver nanoparticles and NH2 ligands.Promising results have been obtained including considerable simplifica-tions in the DNA grafting, a rather homogeneous DNA surface distribu-tion and a reliable and spatially addressed DNA detection. However,intensive optimizations are needed before using this platform for SERSapplications. Further work will concern the morphology of Ag° NPs andtheir distribution on the platform, as well as the effect of these parame-ters on the detection performances.

Acknowledgments

The authors wish to acknowledge Sophie Lecomte from the Centrede Biophysique Moléculaire Numérique (Bordeaux, France) for herhelpful assistance and expertise.

References

[1] S. Pana, J. Xua, Y. Shub, F. Wanga, W. Xiaa, Q. Dingb, T. Xua, C. Zhaoa, M. Zhanga, P.Huanga, S. Lu, Biosens. Bioelectron. 26 (2010) 850.

[2] H. Szmacinski, J.R. Lakowicz, J.M. Catchmark, K. Eid, J.P. Anderson, L. Middendorf,Appl. Spectrosc. 62 (2008) 733.

[3] M. Manesse, R. Sanjines, V. Stambouli, C. Jorel, B. Pelissier, M. Pisarek, R. Boukherroub,S. Szunerits, Langmuir 25 (2009) 8036.

[4] F. Uslu, S. Ingebrandt, D. Mayer, S. Böckert-Meffert, M. Odenthal, A. Offenhauser,Biosens. Bioelectron. 19 (2004) 1723.

[5] J.S. Daniels, N. Pourmand, Electroanalysis 19 (2007) 1239.[6] M. Lee, K.Y. Baik, M. Noah, Y.-K. Kwonn, J.-O. Lee, S. Hong, Lab Chip 9 (2009) 2267.[7] D. Ramos, J. Tamayo, J.Mertens,M. Calleja, L.G. Villanueva, A. Zaballos, Nanotechnology

19 (2008) 1.[8] M. Alvarez, L.M. Lechuga, Analyst 135 (2010) 827.[9] M. Green, F.-M. Liu, L. Cohen, P. Köllensperger, T. Cass, Faraday Discuss. 132

(2006) 269.[10] S. Lecomte, M.-H. Baron, Biospectroscopy 3 (1996) 31.

A C

A732

786

1305 13

25

800 1000 1200 1400 1600

Inte

nsity

(ar

b. u

nits

)

Wavenumber (cm-1)

Fig. 5. Raman spectra of label-free double strand DNA immobilized on photo-patterned Ag°/TiO2 spots (counting time=400 s). The DNA immobilization was performed using thesingle-step procedure (Fig. 1). Main lines are assigned to Adenine and Cytosine Ramanmodes.

2071M. Langlet et al. / Surface Science 605 (2011) 2067–2072

Author's personal copy

[11] K. Faulds, W.E. Smith, D. Graham, Anal. Chem. 76 (2004) 412.[12] S.E.J. Bell, N.M.S. Sirimuthu, J. Am. Chem. Soc. 128 (2006) 15580.[13] S. Rao, S. Raj, S. Balint, C. Bardina-Fons, S. Campoy, M. Llagostera, D. Petrov, Appl.

Phys. Lett. 96 (2010) 213701.[14] Y.W.C. Cao, R. Jin, C.A. Mirkin, Science 297 (2002) 1536.[15] C.S. Thaxton, N.L. Rosi, C.A. Mirkin, MRS Bull. 30 (2005) 375.[16] T. Kang, S. Min Yoo, I. Yoon, S.Y. Lee, B. Kim, Nanoletters 10 (2010) 1189.[17] C.J. Lee, J.S. Kang, M.S. Kim, K.P. Lee, M.S. Lee, Bull. Korean Chem. Soc. 25 (2004)

1211.[18] K.-H. Yang, Y.-C. Liu, C.-C. Yu, Langmuir 26 (2010) 11512.[19] F.-M. Liu, M. Green, J. Mater. Chem. 14 (2004) 1526.[20] C. Yi, C.-W. Li, H. Fu, M. Zhang, S. Qi, N.-B. Wong, S.-T. Lee, M. Yang, Anal. Bioanal.

Chem. 397 (2010) 3143.[21] M.W. Shao, M.-L. Zhang, N.-B. Wong, D. Duo-duo Ma, H. Wang, W. Chen, S.-T. Lee,

Appl. Phys. Lett. 93 (2008) 233118.[22] A. Mills, G. Hill, M. Stewart, D. Graham, W.E. Smith, S. Hodgen, P.J. Halfpenny, K.

Faulds, P. Robertson, Appl. Spectrosc. 58 (2004) 922.[23] Y.-S. Li, X. Lin, Y. Cao, Vib. Spectrosc. 20 (1999) 95.

[24] M. Langlet, S. Permpoon, D. Riassetto, G. Berthomé, E. Pernot, J.C. Joud, J. Photochem.Photobiol. A: Chem. 181 (2006) 203.

[25] S. Briche, Z. Tebby, D. Riassetto, M. Messaoud, E. Gamet, E. Pernot, H. Roussel, O.Dellea, Y. Jourlin, M. Langlet, J. Mater. Sci. 46 (2011) 1474.

[26] D. Riassetto, F. Roussel, L. Rapenne, H. Roussel, O. Chaix, F. Micoud, M. Chatenet,M. Langlet, J. Exp. Nanosci. 5 (2010) 221.

[27] V. Stambouli,M. Labeau, I.Matko, B. Chenevier, O. Renault, C. Guiducci, P. Chaudouët,H. Roussel, D. Nibkin, E. Dupuis, Sens. Actuators B 113 (2006) 1025.

[28] G. Jing, H. Bodiguel, F. Doumenc, E. Sultan, B. Guerrier, Langmuir 26 (2010) 2288.[29] S. Nath, S.K. Ghosh, S. Kundu, S. Praharaj, S. Panigrahi, T. Pal, J. Nanopart. Res.

8 (2006) 111.[30] A. Barhoumi, D. Zhang, F. Tam, N.J. Halas, J. Am. Chem. Soc. 130 (2008) 5523.[31] J.K. Ng, E.S. Selamat, W.-T. Liu, Biosens. Bioelectron. 23 (2008) 803.[32] K.H. Zheng, Y.C. Chou, Y.J. Wu, Y.T. Lee, J. Raman Spectrosc. 41 (2010) 632.[33] K.L. Kelly, E. Coronado, L.L. Zhao, G.C. Schatz, J. Phys. Chem. B 107 (2003) 668.[34] K.G. Stamplecoskie, J.C. Scaiano, V.S. Tiwari, H. Anis, J. Phys. Chem. C 115 (2011)

1403.

2072 M. Langlet et al. / Surface Science 605 (2011) 2067–2072