Universidade de São Paulo

2014-08

On the distinct molecular architectures of

dipping- and spray-LbL films containing lipid

vesicles Materials Science and Engineering C,Lausanne : Elsevier,v. 41, p. 363-371, Aug. 2014http://www.producao.usp.br/handle/BDPI/50421

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Departamento de Física e Ciências Materiais - IFSC/FCM Artigos e Materiais de Revistas Científicas - IFSC/FCM

On the distinct molecular architectures of dipping- and spray-LbL filmscontaining lipid vesicles

Pedro H.B. Aoki a,⁎, Priscila Alessio a, Diogo Volpati b, Fernando V. Paulovich c, Antonio Riul Jr. d,Osvaldo N. Oliveira Jr. b, Carlos J.L. Constantino a

a DFQB, Faculdade de Ciências e Tecnologia, UNESP Univ Estadual Paulista, Presidente Prudente, SP 19060-900, Brazilb São Carlos Institute of Physics, University of São Paulo, CP 369, São Carlos, SP 13560-970, Brazilc Institute of Mathematical and Computer Sciences, University of São Paulo, CP 668, São Carlos, SP 13560-970, Brazild Applied Physics Department, Gleb Wataghin Institute of Physics, State University of Campinas, UNICAMP, Campinas, SP 13083-859, Brazil

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

Article history:Received 7 February 2014Received in revised form 2 April 2014Accepted 26 April 2014Available online 5 May 2014

Keywords:Layer-by-layer filmsMolecular architectureSensingInformation visualizationErythrosin

The introductionof sprayingprocedures to fabricate layer-by-layer (LbL)filmshasbroughtnewpossibilities for the con-trol of molecular architectures and for making the LbL technique compliant with industrial processes. In this study weshow that significantly distinct architectures are produced for dipping and spray-LbL films of the same components,which included DODAB/DPPG vesicles. The films differed notably in their thickness and stratified nature. The electricalresponseof the two types offilms to aqueous solutions containingerythrosinwas also different.Withmultidimensionalprojectionsweshowed that the impedance for theDODAB/DPPGspray-LbLfilm ismore sensitive to changes in concen-tration, being thereforemorepromising as sensingunits. Furthermore,with surface-enhancedRaman scattering (SERS)we could ascribe the high sensitivity of the LbL films to adsorption of erythrosin.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The control of molecular architecture is crucial for many applica-tions of organic films, particularly because synergy may be soughtupon combining distinct materials in the same film [1,2], beingalso a key feature of the layer-by-layer (LbL) technique [3,4]. Thesynergy achieved in LbL films has been extensively exploited in var-ious applications, including sensing and biosensing [1,5], as themild conditions are required for film fabrication and the retentionof entrapped water are essential for the successful immobilizationof biomolecules [5,6]. For instance, judicious combinations of dis-tinct materials for electronic tongues (e-tongues) [1,5,7,8] haveallowed detection of analytes down to nanomolar concentrations orbelow [9,10], in addition to the diagnosis of diseases [11–14] and infood control [15]. In many such cases, altering the order at which thedifferent materials are adsorbed or even the number of layers affectsconsiderably the film response [5,9,16].

The recent variation of the LbL technique based on spraying [17–22]has brought new possibilities, since the fast procedure makes it probablycompliant with industrial processes. With spraying one may obviate onepractical limitation of dipping LbL films, which require long periods oftime for complete layer adsorption and normally coat small areas.

Spraying is claimed to speed up the LbL process up to 500 times due tothe immediate contact between the surface and the approaching sprayedmaterial [18]. Spray-LbLfilms have been used for fabricating electrodes inhigh power and energy devices [23], solar cells [24], double-layer anti-reflection films [25], cotton fibers for flame retardancy [26], depositionof colloidal nanoparticles [27,28], polysaccharides [29] and conductingpolymers [30,31].

An additional feature of the spray-LbL films is that the moleculararchitecture may differ from the corresponding dipping-LbL films[32], which calls for further research both in terms of film character-ization as well as in the identification of novel applications. In thispaper, we build upon a previous work [32] where dipped- andspray-LbL films containing phospholipid vesicles were compared,and investigate the molecular architectures of DODAB/DPPG LbLfilms. Using a combination of experimental methods, we shall showthat the molecular architecture of spray-LbL films differs consider-ably from that of dipping LbL films. Because it is known that sensingunits built with the same materials but different processing tech-niques may display different electrical responses, we correlate thetype of LbL film with the ability to detect trace amounts of xanthenederivative erythrosin in water through impedance spectroscopymeasurements. Our interest in the dye erythrosin arises from its ver-satile use, spanning from lasing action [33], photodynamic therapy(PDT) [34] to food industry [35,36], especially because in the latterits waste represents an environmental issue.

Materials Science and Engineering C 41 (2014) 363–371

⁎ Corresponding author. Tel.: +55 18 32295876.E-mail address: pedroaoki@gmail.com (P.H.B. Aoki).

http://dx.doi.org/10.1016/j.msec.2014.04.0670928-4931/© 2014 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Materials Science and Engineering C

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

2. Experimental section

2.1. Reagents and solutions

The anionic phospholipid DPPG [1,2-dipalmitoil-sn-3-glicero-fosfo-rac-(1-glycerol)] was purchased from Genzyme Pharmaceuti-cals. The cationic lipid DODAB (dioctadecyldimethylammoniumbromide), the anionic poly(sodium 4-styrenesulfonate) (PSS), thecationic poly(allylamine hydrochloride) (PAH) and xanthene eryth-rosin, were acquired from Sigma-Aldrich Co. All chemicals wereused without further purification. The molecular weights of DPPG,DODAB, PAH, PSS and erythrosin are 744.95; 630.95; 15,000;70,000; and 835.89 g/mol, respectively, and their molecular structuresare shown in Fig. 1. The spray-LbL films were fabricated from DPPGand DODAB aqueous dispersion using ultrapure water (18.2 MΩ·cmand pH 5.6) obtained from a Milli-Q system, model Simplicity. TheDPPG dispersion at 0.74 mg/mL (1.0 mM) was prepared by addingultrapure water at room temperature (22 °C) to the powder, undergentle stirring. The 0.63 mg/mL (1.0 mM) concentration of DODABdispersion was prepared following the methodology by Feitosaet al. [37]: DODAB powder was added to ultrapure water and themixture was heated under stirring to 60 °C (above DODAB fusiontemperature, Tm≈ 45 °C) for a few minutes. Then, the DODAB disper-sion was cooled to room temperature (22 °C). The cast films reportedhere were prepared by dropping the desired solution (ca. 1 mL) ontothe substrate, which was left to dry under room temperature.

2.2. Layer-by-layer film fabrication

2.2.1. Dipping-LbL filmsThe DODAB/DPPG dipping-LbL films were grown upon adapting

the experimental procedure reported in [38]. The substrate wasimmersed into distinct solutions according to the followingsequence: DODAB dispersion (3 min) → ultrapure water gentlystirred to remove non-adsorbed DODAB (1 min) → DPPG disper-sion (3 min) → ultrapure water to remove non-adsorbed DPPG(1 min). With this procedure the first DODAB/DPPG bilayer wasformed, and multilayers were obtained by repeating this four-step sequence.

2.2.2. Spray-LbL filmsThe deposition of (DODAB/DPPG)n spray-LbL films was performed

by spraying DODAB and DPPG dispersions alternately using the ex-perimental setup described in ref. [32], which consisted of an air-brush spray, model BD-123A from LEEpro tolls with a nozzle of 0.3 mmdiameter and 7 mL of fluid cup capacity. For spraying deposition, a per-pendicular orientation was adopted between the spray axis and thecoating surface, being 30 cm apart from each other. The airbrush sprayswere filled with DODAB and DPPG dispersions alternately using a con-stant pressure of 10 psi during 2 s. A valve regulator was used to controlthe spraying pressure from the compressor. These parameters werechosen after testing different combinations involving spraying pressures,spraying times, with and without rinsing steps [32]. The final setupallows for spraying solutions to form a thin layer on the desired surface,which dries within a few seconds.

The number of spraying (or dipping) bilayers and the type of sub-strate were chosen according to the characterization technique. Forinstance, films were grown onto quartz plates for UV–Vis extinctionspectroscopy, onto AT-cut quartz crystal coated with Au plates forquartz crystal microbalance (QCM) measurements, onto Ge for Fou-rier transform infrared spectroscopy (FTIR), onto glass thermallytreated (600 °C for 2 h) for atomic force microscopy (AFM), andonto interdigitated Pt electrodes for impedance spectroscopy. Thesubstrates were previously cleaned using neutral detergent, beingsequentially and extensively washed in distilled water, acetone(5 min sonication), chloroform (5 min sonication), and ultrapurewater. This procedurewas sufficient to prepare substrate surfaces with-out drastic treatments such as with the use of piranha solution.

2.3. Characterization techniques

The growth of LbL films (spray and dipping) was monitored via UV–Vis extinction spectroscopy using a Varian spectrophotometer; modelCary 50, from 190 to 1100 nm, and via mass adsorption using a quartzcrystal microbalance Stanford Research Systems Inc., model QCM 200.Shifts in frequency were continuously monitored with time and themass gain (Δm, in grams) was obtained using the Sauerbrey equa-tion [39]: ΔF = (−2.3 × 106 F02Δm)/A, where ΔF is the frequency shift(in Hz), F0 is the resonance frequency (without coating, in MHz), andA is the electrode area (0.4 cm2). The FTIR spectra were taken using aBruker spectrometer, model Vector 22, equipped with a DTGS detector.The morphology was characterized through AFM using a Nanosurf In-strument model easyScan 2, with a tip of silicon nitride and using thetapping mode. All images were collected with high resolution (512lines per scan) at a scan rate of 0.5 Hz. The images were processedwith the software Gwyddion 2.19. The micro-Raman scattering experi-ments were conducted with a micro-Raman Renishaw spectrograph,model inVia, with laser excitation at 633 nm. The system is equippedwith a Leica microscope, whose 50× (NA 0.75) objective lens allowsfor collecting the spectra with ca. 1 μm2 spatial resolution. Single-point spectra were recorded with 4 cm−1 resolution and 10 s accumu-lation time, whereas two dimensional (2D) mapping results were col-lected through the rastering mode by using a computer-controlledmotorized stage (XY).

Impedance spectroscopy measurements were carried out with aSolartron 1260A impedance analyzer. The sensing array comprised 6sensing units: a bare Pt interdigitated electrode and Pt interdigitatedelectrodes coated with 5 bilayers of the following LbL films: dippingPAH/PSS, spray PAH/DPPG, dipping PAH/DPPG, spray DODAB/DPPGand dipping DODAB/DPPG. The bare Pt electrode was used to monitorany change in the electrical response caused by ultrathin films. The de-tails of fabrication of dipping and spray-LbL films of PAH/DPPG can befound in ref. [32]. Impedance spectra were acquired from 1 Hz to1 MHz using 50 mV of amplitude, with the data analyzed in terms ofreal capacitance. Three consecutive measurements were performedwith sensing units immersed into ultrapure water (18.2 MΩ·cm)Fig. 1.Molecular structures of PAH (mer), PSS (mer), DPPG, DODAB and erythrosin.

364 P.H.B. Aoki et al. / Materials Science and Engineering C 41 (2014) 363–371

used as reference and into erythrosin aqueous solutions at 10−9, 10−7,10−6,10−5 and 10−4 M. The course of measurements starts with ultra-purewater, and then from the lowest (10−9M) to the highest (10−4M)erythrosin concentrations. Between each set of measurements involv-ing a specific concentration of erythrosin, the sensing units were care-fully washed with ultrapure water. The sensing units were left soakedat 20 min inside the solutions before data acquisition to enable a stablereading [40].

2.4. Data analysis with multidimensional projections

In recent years several innovations have been implemented insensing and biosensing, since researchers increasingly obtain largeamounts of data in their experiments, as it is discussed in Oliveiraet al. [7]. Among themethods used for analysis are the so-called mul-tidimensional projections, the most widely used being PrincipalComponent Analysis (PCA) [41]. PCA is a multivariate statisticalanalysis employed to find patterns in data [41]. In this paper, the im-pedance spectroscopy data for the various sensing units immersedinto aqueous solutions with different concentrations of erythrosinwere treated with PCA and another multidimensional projectiontechnique, referred to as Sammon's mapping [42]. In these methodsdata from the original space – in our case the impedance (or capacitance)vs. frequency curve – are mapped onto a 2D plot in an attempt to placedata points from similar samples close to each other. For Sammon'smap-ping, this is performedwith an optimization procedure, as follows. Let thedata set be represented by X = {x1,x2,…,xn}, with δ(xi, xj) being the dis-similarity (distance) between two data instances i and j. The graphicalmarkers corresponding to X projected on the plane are represented byY = {y1,y2,…,yn}, determined through an injective function f: X → Ythat minimizes |δ(xi, xj) − d(f(xi),f(yj))| ≈ 0, ∀xi, xj ∈ X [43], where

d(yi,yj) is the distance function on the projected plane. For Sammon'smapping, the error function used in the minimization is given as:

SSam ¼ 1Xib j

δ xi; xj

� �X d yi; yj

� �−δ xi; xj

� �� �2

δ xi; xj

� �

where δ and d are the distance functions defined above.

3. Results and discussion

3.1. Monitoring the growth of DODAB/DPPG dipping- and spray-LbL films

A similar, linear growthwas observed for the dipping- and spray-LbLfilms of (DODAB/DPPG)n, according to the UV–Vis extinction andQCM data in Fig. 2. The average mass deposited per bilayer is higherfor the spray-LbLmethod (0.28 μg) than using the dipping-LbLmeth-od (0.17 μg), though the absorbance and mass are slightly higher fordipping-LbL films, since they are deposited on both sides of the sub-strate, in contrast to the deposition on only one side for the spray LbLfilms. The more efficient adsorption for the spray-LbL method is consis-tent with previous work for dipping- and spray-LbL films of PAH/DPPG[32]. The details of the QCM data for monitoring LbL film growth aregiven in Fig. S1 in the Supporting Information.

FTIR absorption spectroscopy was used to investigate possible in-teractions between DPPG and DODAB layers, which may govern filmgrowth. The FTIR spectra are shown in Fig. S2, with the main assign-ments [44–47] given in Table S1 of the Supporting Information.Overall, the bands for the spray- and dipping-LbL films are practicallya simple superposition of the spectra from DODAB and DPPG castfilms. The small differences between cast and LbL films may indicate

Fig. 2.UV–Vis extinction spectra for the LbL films ofDODAB/DPPG: (a) dipping- and (b) spray-LbLfilms deposited up to 12bilayers. The linear dependence of (c) absorbance at 200 nmand(d)mass (QCMmeasurements) as a function of DODAB/DPPG bilayers for dipping- and spray-LbL films. For the spray-LbL films the deposition was performed on only one side of the sub-strate, in contrast to the dipping-LbL films that were deposited on both sides.

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weak interaction between DODAB and DPPG. Therefore, electrostaticinteractions between CH3-N+ (DODAB) and PO4

− (DPPG) groups ap-pear not to be the main driving force to grow DODAB/DPPG dipping-and spray-LbL films. This is in contrast to the results for PAH/DPPGLbL films [38], where stronger interactions between DPPG and PAHleading to larger FTIR spectral changes were found for dipping-LbLfilms than for the spray-LbL films.

For DODAB/DPPG, a shoulder at 1717 cm−1 was observed only for thespray-LbL film, and may be assigned to hydrogen bonds between COOester groups andglycerolOHgroupsof adjacentDPPGmolecules, accordingto Dicko et al. [45] who reported a shoulder at 1720 cm−1 in the spectrumof a DPPG Langmuir monolayer using polarization-modulated infraredreflection absorption spectroscopy (PM-IRRAS). Dicko et al. [45] arguedthat hydrogen bonding was favored at low surface pressures due to thehigher disorder in the DPPG Langmuir film, and therefore one mayhypothesize that aggregates of vesicles in DODAB/DPPG spray-LbLfilms could lead to a larger disorder. The latter was observed by AFMand surface-enhanced Raman scattering (SERS)which are discussed next.

In order to complement the discussion on effects from film architec-tures, in subsidiary experimentswith distinct lipids and polyelectrolytes,we observed that spray-LbL films can be grown from either oppositely orequally chargedmaterials, while dipping-LbLfilms could not be obtainedfor equally chargedmaterials or with only a single component. In case ofzwitterionic phospholipid, the growth of PAH/DPPC dipping-LbL film islimited to 13 bilayers. [38] Spray-LbL films containing multilayers ofPAH(+)/DPPG(−), DODAB(+)/DPPG(−), PAH(+)/DPPC (zwiterionic)or PAH(+)/DODAB(+) were successfully grown, as displayed inFig. S3a. For the oppositely charged materials, the amount of massdeposited per bilayer is actually larger than the sum of the masses forindividual layers in films grown using only one component in Fig. S3b.This is indicated in Table S2, where the mass deposited per bilayer ofPAH/DPPG and DODAB/DPPG was ca. 20% and 30% higher than thesum (PAH + DPPG and DODAB + DPPG), respectively. Also shown inTable S2 is that for the film containing the zwitterionic DPPC, themass deposited per bilayer was almost the same as the sum of the indi-vidual layers (PAH + DPPC). As for the spray-LbL film obtained withequally charged materials, which is demonstration itself that electro-static interactionsdonot governfilm growth, themass deposited per bi-layer is ca. 20% less than the sum of individual layers (PAH + DODAB).Note also that Table S2 only brings data for spray-LbL films sincedipping-LbL films could not be built with a single component. In sum-mary, these results indicate that electrostatic interactions do play a

role but are not the only relevant interaction for building the spray-LbL films studied here. Furthermore, the forces driving spray- anddipping-LbL films of the same materials may differ. The importance ofmolecular architecture is also clear by comparingwith data from the lit-erature. For example, Porcel et al. [48] reported that for poly-glutamicacid (PGA) and PAH spray-LbL films could not be grown when onlyone solution (polyanion or polycation) was sprayed onto the substrate.The latter experiment was performed in different experimental condi-tions than reported here, generating a molecular architecture whereelectrostatic interaction is crucial for the film growth.

3.2. The architecture of dipping- and spray-LbL films

The structuring of LbL films has been studied formany years, with is-sues such as interpenetration of layers and stratification being probedwith neutron scattering and X-ray reflectivity measurements [49–51].Here we used AFM to investigate the morphology of dipping- andspray-LbL DODAB/DPPG films. The AFM images in Fig. 3a and b for 10-bilayer dipping- and spray-LbL films indicate that most of the vesicleswere not destroyed under the experimental conditions used. The pres-ence of DODAB and DPPG vesicles is evidenced by the spherical-likestructures, with diameters ranging from 50 to 250 nm for dipping-andfrom 50 to 140 nm for spray-LbL films. According to the profiles inFig. 3c–d and the histograms with the diameter distributions inFig. 3d–e, the average size of the vesicles is ca. 200 nm and 100 nm fordipping- and spray-LbLfilms, respectively.We concluded thatmost ves-icles were preserved based not only on these AFM images but also onprevious evidence from the literature that the vesiclesmaintain their in-ternal aqueous phase in LbL films [38,52]. Some of the vesiclesmay nev-ertheless have been either fused or destroyed by the spraying pressure,as less vesicles are found in the spray-LbL film. The shape of the vesicleswas affected considerably for the spray-LbL film, with a decrease of ap-proximately one order of magnitude in the maximum height in Fig. 3cand d, being 3–6 nm for the spray-LbL films (several vesicles were in-vestigated). The heightmeasured here does not coincidewith the radiusof the vesicle, probably because the vesicles can be embedded into thefilm and the profiles measured are above the vesicle equator. Neverthe-less, due to the pressure applied during spraying, the vesicles might bestructured as flattened spheroids, as depicted in the cartoons in Fig. 4with spherical-like structures in dipping-LbL films and flattened spher-oids in spray-LbL films. For the same reason, the root mean square(RMS) roughness decreased from 8.3 nm for dipping-LbL films to 0.6

Fig. 3. AFM topographic images in 2 and 3 dimensions for 10-bilayer DODAB/DPPG: (a) dipping- and (b) spray-LbL film. AFM vesicle profiles recorded for (c) dipping- and (d) spray-LbLfilms. Histograms for diameter distribution (nm) of vesicles found in (e) dipping- and (f) spray-LbL film.

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nm for spray-LbL films, for areas with the same size. Such differences inmorphology between dipping- and spray-LbL films should be expectedfrom the literature [18,21].

The AFM images suggest that the DODAB and DPPG structures arenot deposited in stratified layers in spray-LbL films, in contrast to thedipping-LbL films, and this is also depicted in Fig. 4.

Further evidence for the stratification (or not) of the multilayersin the LbL films is obtained from SERS for which customized architec-tures were built. Based on our previous experience with similar LbLfilms [32,53], we fabricated tetralayers with different supramolecu-lar architectures, with one layer containing a reporter moleculewhose SERS signal could be distinguished from contributions of theother layers. The reporter molecule used was the xanthene erythro-sin, which has high cross section for SERS [54,55], and was chosenbecause we also used it in sensing experiments to be discussedlater on. To achieve SERS, one of the deposited layers contained col-loidal silver nanoparticles (AgNPs). Dipping- and spray-LbL filmswere built with tetralayers in two sequences: DODAB/DPPG/AgNPs/erythrosin and DODAB/AgNPs/DPPG/erythrosin. If the films are strat-ified, the SERS effect would be observable for erythrosin only in the firstsequence, since in the latter sequence the distance from the AgNPswiththe DPPG spacing layer would hamper SERS. Conversely, if SERS isobtained for the second sequence, then the tetralayer would not bestratified.

In control experiments, we observed SERS in DODAB/DPPG/AgNPs/erythrosin tetralayers for both dipping- and spray-LbL films, since the

erythrosin layer was adjacent to the AgNPs (results not shown). ForDODAB/AgNPs/DPPG/erythrosin tetralayer, however, Fig. 5a showsthat the SERS effect was only obtained for the spray-LbL film. For thedipping-LbL film, the signal was very weak, corresponding to the con-ventional Raman scattering. The signal could nevertheless be assignedto erythrosin by the similarity in the peaks with the SERS spectrum, asindicated in the magnified spectrum in Fig. 5a, marked with RS and anarrow. Therefore, for the dipping-LbL film, DODAB and DPPG vesiclesare indeed deposited in stratified layers, as indicated in the cartoon inFig. 4. In contrast, the cartoon for the spray-LbL film depicts non-stratified layers, consistent with the AFM images in Fig. 3. Fig. 5b dis-plays the molecular structures proposed for dipping- and spray-LbLfilms. Note that because the tetralayers are not stratified in the spray-LbL film, the same architecture could in principle be obtained withspraying mixtures of the components of the 4 layers.

The inset in Fig. 5a shows a Raman mapping built up by collectingspectra point-by-point along an area of 80 μm × 80 μm with 2 μmsteps, leading to a total of 1681 spectra recorded for the tetralayerdeposited by spraying. The intensity of the band at 1500 cm−1

(C_C stretching) was plotted along the mapped area where brighterspots refer to higher intensities. This band intensity varies signifi-cantly from spot to spot owing to the spatial distribution of AgNPs(the closer erythrosin-AgNPs, the stronger the SERS signal). Thereare a few dark spots where the DPPG vesicles might be positionedbetween erythrosin and AgNPs, suppressing the SERS signal, leadingto conventional Raman spectra. An estimation of the enhancement

Fig. 4. Possible supramolecular architectures in dipping- and spray-LbL films of DODAB/DPPG. (a) Dipping-LbL films: stratified layers with DODAB and DPPG forming spherical-like struc-tures. (b) Spray-LbL films: non-stratified layers with DODAB and DPPG forming flattened spheroid structures.

Fig. 5. (a) Conventional Raman and erythrosin SERS spectra collected for dipping- and spray-LbL films, respectively, both containing 1-tetralayer of DODAB/AgNPs/DPPG/erythrosin. Theinset shows the Ramanmapping area built by collecting spectra point-by-point along an area of 80 μm× 80 μmwith a step of 2 μm(total of 1681 spectra) and plotting the intensity of theband at 1500 cm−1. (b) Molecular structures proposed for dipping- and spray-LbL films.

367P.H.B. Aoki et al. / Materials Science and Engineering C 41 (2014) 363–371

factor can be made by calculating the intensity ratio between a brightand a dark spot [53], which led to a factor of 104. This is in good agree-ment with enhancement factors for SERS due to the electromagneticmechanism, i.e., the enhancement is basically produced by the electro-magnetic field amplified surrounding themetal nanoparticle surface in-stead of changes in the erythrosin polarizability [56]. The latter issupported by the similarity (wavenumbers and relative intensity pro-file) between the erythrosin Raman spectrum (scaled to fit the plotfor an easier visualization) and the erythrosin SERS spectrum. This indi-cates that erythrosin does not form a complex with AgNPs, i.e. there isphysisorption with no chemical interaction.

3.3. Sensing erythrosin

Erythrosin in aqueous solutions could be detected by sensing unitsmade with DODAB/DPPG dipping- and spray-LbL films deposited ontoPt interdigitated electrodes. For the sake of comparison, bare Pt elec-trodes and electrodes coated with PAH/PSS and PAH/DPPG were alsoused. Because the electrical properties of solid/liquid interfaces arehighly sensitive to any change in the liquid, the incorporation of increas-ing amounts of erythrosin caused the capacitance vs. frequency curvesto be altered, as indicated in Fig. S4 in the Supporting Information.

A visual inspection of the data in Fig. S4 points to distinct abilities todetect erythrosin for the different sensing units, and this can be visual-ized more clearly in the projection using Sammon's mapping [42] inFig. 6, where only the data for the lowest concentrations are shown be-cause they are themost demanding to distinguish. Significantly, the unitcontainingDODAB/DPPG spray-LbLfilm is far superior to the other ones,since the distances among the clusters of different erythrosin concen-trations were much larger than for the other sensing units.

The differences resulting from the distinct molecular architecturesare corroborated by the PCA plots obtained with the capacitance takenat a fixed frequency for each sensing unit (1 kHz), similarly to previousworks [10,32,52,57]. Fig. 7a shows a PCA plot where the combined re-sponse of the sensingunits forming a sensor array can distinguish eryth-rosin solutions down to 1 nM. In order to assess the importance of eachsensing unit for the overall high sensitivity, an empirical test was madein which PCA plots were obtained by eliminating one of the sensingunits [38,52,57]. The plots were slightly affected when the excluded

sensing unit was the Pt bare electrode or the Pt electrode coated withPAH/DPPG, PAH/PSS and DODAB/DPPG dipping LbL films (results notshown). However, the PCA plots were significantly affected by remov-ing the DODAB/DPPG spray-LbL film, as shown in Fig. 7b, followed bythe PAH/DPPG spray-LbL film (result not shown). Indeed, more than99% of the data are explained by PC1 and PC2 when the sensing unitformed by DODAB/DPPG spray-LbL film is removed. This increase inthe correlation between PC1 and PC2, in relation to PCA plots obtainedconsidering all sensing units (Fig. 7a), indicates that the sensing unitcomposed by DODAB/DPPG spray-LbL film presents a higher dispersionof capacitance data in relation to the other sensing units. The latter is inagreement with the loading plots exhibited in Fig. 7c and d, which dis-play the sensing units into a 2D graph according to their similarities.The closer the sensing units in the loading plot the higher the similarityamong them in the impedance data when immersed in liquid samples[10,32]. It is seen that the sensing units composed by DODAB/DPPGfollowed by PAH/DPPG spray-LbL films are the most different sensingunits, in relation to the others. Besides, there is a trend in which thesensing units are grouped according to the technique employed infilm fabrication (dipping or spray-LbL films), highlighting the effect ofthe supramolecular architecture. Fig. 7d shows a zoomed regionwhere all sensing units composed by dipping-LbL films are broughttogether.

A further way to analyze the differences between the two types ofarchitecture in dipping- and spray-LbLfilms is tomodel the electrical re-sponse of the sensing units using a parallel R-C circuit and to obtain theNyquist diagrams (Z′ vs. Z″) [58]. Fig. 8 shows the plots for the DODAB/DPPG spray (a) and dipping (b) sensing units exposed to the highestconcentrations where the resistance varied almost linearly with theerythrosin concentration. Significantly, while Z′ (i.e. the resistance) in-creased with increasing erythrosin concentration for the spray-LbLfilm, the opposite occurred for the dipping-LbL film.

The response of the several sensing units could be combined as in ane-tongue system as indicated in the results in Fig. 7, where distinctconcentrations could be easily distinguished, similarly to the resultsin Fig. 6. However, in control experiments we noted that the imped-ance spectrum for pure water after a series of experiments did notreturn to the original one. This means that the films were affectedby erythrosin in a way that even several washing procedures were

Fig. 6. Sammon's mapping plot of the impedancemeasurements obtained from the distinct sensing units, whichmay be identified by the colors in the inset. No axes are shown in the plotsince what matters in this type of projection is the relative distance between data points, with closer points being associated with more similar samples.

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not sufficient to clean the electrodes. Therefore, the data displayed inFigs. 6 or 7 cannot be used to estimate the detection limit, for exper-iments will have to be performed with different sensing units foreach erythrosin concentration.

The reason for the irreversible changes in the electrical response ofthe sensing units is probably adsorption of erythrosin molecules, simi-larly to reports in the literature for other analytes [10,32,38,52,59].This was confirmedwithmicro-Ramanmapping experiments taken be-fore and after the units were exposed to erythrosinsolutions. Domainsat the film surfaces could be found in all sensing units, as shown bythe opticalmicroscopy in Fig. 9a for the DODAB/DPPG spray-LbL sensingunit. The chemical identification of the domains could be made bysuperimposing a Raman mapping to the optical microscopy in Fig. 9b.

The point-by-point mapping was obtained by collecting Raman spectrain an area of 80 μm· 80 μmwith a step of 2 μm, leading to a total of 1681spectra. The intensity of the band at 1500 cm−1 (C_C stretching) wasplotted and brighter spots refer to more intense Raman signal.

The Raman spectra collected are attributed to erythrosin [60,61],consistent with the spectrum for erythrosin powder in Fig. 9c. It shouldbe noted that DODAB and DPPG possess low cross sections for Ramanscattering using the 633 laser line and do not exhibit Raman signal forthese thin films [32,38,53,62]. The intensity of the Raman band at ca.1500 cm−1 for the erythrosin micrometer domain follows the domainspatial distribution according to the optical image, i.e., the Raman signalis stronger where the erythrosin domain is clearly seen in the opticalimage. Therefore, thismorphological change on thefilm surface induced

Fig. 7. PCA plots with capacitance at 1 kHz for (a) all sensing units and (b) excluding the (DODAB/DPPG)5 spray-LbL sensing unit. (c) loadings for PC1 vs PC2 and (d) zoom of the regioncirculated by the dashed line.

Fig. 8. Nyquist diagram with experimental and fitting (parallel R-C circuit) results for DODAB/DPPG spray (a) and dipping (b) sensing units.

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by adsorption of erythrosin molecules is responsible both for the highsensitivity reached by the sensing units and the irreversible changes inthe electrical response.

4. Conclusion

The LbL films of DODAB/DPPG vesicles produced by conventionaldipping and by spray exhibit distinct architectures. The vesicles areflattened on spray-LbL films, which are not properly stratified,while stratified LbL films are obtained with dipping. Such differencesin architecture were also reflected on the electrical response of theLbL films deposited onto Pt interdigitated electrodes when im-mersed into aqueous solutions of erythrosin. The sensing units arehighly sensitive to trace amounts of erythrosin, probably becausethe latter are adsorbed on the film surface as demonstrated. A furtherpossibility to be exploited is to use SERS, as the results discussed herepoint to a high sensitivity that can be achieved with suitable substrates.

Acknowledgments

Thisworkwas supported by FAPESP, CNPq and the nBioNet networkfrom CAPES (Brazil).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.msec.2014.04.067.

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