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Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 288– 293

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

journa l h omepa g e: www.elsev ier .com/ locate /co lsur fa

luorescence enhancement for the complex PAMAM–BSA in the presence ofhotonic crystal heterostructures

lexandra Mocanua,∗, Bogdan Marculescua, Raluca Somoghib, Florin Miculescuc,ristian Boscorneaa, Izabela Cristina Stancua

University Politehnica of Bucharest, Department of Polymer Science, 149 Calea Victoriei, RO-010072 Bucharest, RomaniaNational Research and Development Institute for Chemistry and Petrochemistry – ICECHIM, 202 Independence Street, District 6, CP 35-174, 060021 Bucharest, RomaniaUniversity Politehnica of Bucharest, 313 Spl. Independentei, RO-74204 Bucharest, Romania

r t i c l e i n f o

rticle history:eceived 8 July 2011eceived in revised form9 September 2011ccepted 5 October 2011vailable online 12 October 2011

a b s t r a c t

The paper presents the optical characterization of the multilayer film composed of styrene(ST)–poly(ethylene glycol) methyl ether methacrylate (PEGMA 1100)–gold nanoparticles(Au)–poly(amidoamine) PAMAM (G4)–bovine serum albumin (BSA). The addition of the last layercomposed of BSA resulted in an unusual optical behaviour, i.e. increase of the fluorescence emissionintensity, respectively the intensity of the UV–vis reflection, compared with the ST–PEGMA 1100–Au–G4film. The explanation could be attributed to the presence of photonic crystal heterostructures. The

eywords:hotonic crystalsold nanoparticleserum albuminAMAM

multilayer film has been characterized by optical microscopy, AFM, UV–vis, and fluorescence.© 2011 Elsevier B.V. All rights reserved.

luorescenceV–vis

. Introduction

Serum albumin is one of the most extensively studied proteinsor many years. It is the most abundant protein in blood plasmaith a typical concentration of 50 g/L and functions as a transportrotein for numerous endogenous and exogenous substances. Itlso plays an important role in regulating the colloid osmotic pres-ure of blood. It provides about 80% of the osmotic pressure and isesponsible for the pH maintenance in blood [1]. Many researchersave studied the structures, functions and properties of serum albu-ins to understand their interactions with other molecules and

igands. Serum albumin can interact with dendrimers [2–4], whichre relatively new class of globular polymers. They possess a centralore, branches expanding from the core and many terminal groups5,6].

The full generations of PAMAM dendrimers have hydroxyl ormino groups on their surfaces whereas the half generations havearboxyl or sodium carboxyl groups. The abundance of different

unctional surfaces add dendrimers numerous unique properties,.g. aqueous solubility and stimuli (temperature, pH, etc.) responsi-ility [7,8]. These properties make dendrimers favourable in many

∗ Corresponding author. Tel.: +40 0740004385.E-mail address: mocanu.alexandra53@yahoo.com (A. Mocanu).

927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2011.10.006

biomedical and pharmaceutical areas such as bio-imaging, tissueengineering, drug and gene delivery [9–12]. By far (PAMAM) den-drimer is the most intensively investigated [13–19].

The effect of dendrimers on bovine serum albumin (BSA) con-sists in a decrease/quenching of the fluorescence of the twotryptophan residues contained in BSA [20–24]. The possible rea-son for the decreasing fluorescence intensity is the electrostaticdendrimer–protein interactions (Scheme 1).

Metal nanoparticles can be used as physical support forBSA–PAMAM complex, whereas gold nanoparticles (Au)–PAMAMnanocomposites are used as biosensors [24,25], quantum dots [26],and drug delivery systems [27]. Au nanoparticles can be used asa quencher to decrease the fluorescence intensity of fluorophores[28–30]. The dynamic quenching process included an instanta-neous exciplex formed between excited fluorescent molecules andquenchers [31]. These exciplexes could not emit fluorescence orbecome different from the original fluorescent molecules, whichcause quenching to happen. In the process of resonance energytransfer, the efficiency depends on the overlap degree of emissionspectra of the donor and the absorption spectra of the acceptor. Thehigher the overlap, the higher the efficiency [24]. The prominent

overlap between the absorption spectrum of Au and the emis-sion spectrum of PAMAM provides increased probability of energytransfer from the excited dendrimer to Au, and hence the intensequenching of the fluorescence (Scheme 2).

A. Mocanu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 288– 293 289

–naatotohsoptgloefitol

BhP

2

2

t1s

Scheme 1. PAMAM–BSA complex.

Photonic crystals (PCs) constitute a fascinating class of materialspromising candidates for nanoscale optoelectronic devices for theext generation information technology [32–34]. They are gener-lly characterized by artificial structures with a periodic dielectricrrangement which does not allow propagation of light in all direc-ions for a given frequency range. This phenomenon induces thepening of photonic stop-bands or band gaps due to Bragg diffrac-ion [35,36]. The stop-band of the PCs represents the narrow rangef specific wavelengths in which the propagation of light is pro-ibited. In this context, the synthesis of monodisperse colloidalpheres with sub-micronic diameters has lately attracted a lotf interest from various researchers, due to their self-assemblingroperties leading to crystalline structures of synthetic opal afterhe removal of the dispersion medium. Crystalline lattices of inor-anic or polymer particles have a highly ordered structure thateads to PCs properties. One of the most promising methods ofbtaining monodisperse colloidal polymer particles is the soap-freemulsion polymerization [37–41]. Various methods for obtaininglms of colloidal particles, such as gravitational sedimentation, cen-rifugation, vertical deposition, physical confinement, interfacialr electric field induced self-assembly have been described in theiterature [42–48].

In this work, a multilayer film composed of PCs, Au, PAMAM andSA was obtained (Scheme 3). The optical influence of PCs substrateas been investigated, given that Au decreases the fluorescence ofAMAM and PAMAM also decreases the BSA fluorescence.

. Materials and methods

.1. Materials

Styrene (ST) (Merck) was purified through vacuum distilla-

ion. Poly(ethylene glycol) methyl ether methacrylate (PEGMA100) (Aldrich) was used without purification. Potassium per-ulphate (KPS) (Merck) was recrystallised from an ethanol/water

Scheme 2. PAMAM–Au complex.

Scheme 3. PCs–Au–PAMAM–BSA complex, multilayer film.

mixture and then vacuum dried. Tetrachloroauric acid trihy-drate 99.5% (HAuCl4·3H2O) (Merck), trisodium citrate dihydrate(Na3C6O7·2H2O) (Fluka), PAMAM (G4) (Aldrich), BSA (Fluka) wereused as received.

2.2. Soap-free emulsion polymerization

6.5 ml ST and 0.25 ml PEGMA 1100 were added in 100 ml dis-tilled water together with 0.0625 g KPS. The reaction mixture wasnitrogen purged and then maintained for 8 h at 75◦ C under contin-uous stirring. The final dispersion was dialyzed in distilled water for7 days, using cellulose dialysis membranes (molecular weight cut-off: 12,000–14,000), in order to remove the unreacted monomerand initiator.

2.3. Preparation of gold (Au) nanoparticles

Au colloids were prepared by Na3C6O7·2H2O reduction ofHAuCl4. 90 ml HAuCl4 3 × 10−4 M aqueous solution was allowedto boil and then 3.6 ml Na3C6O7 6.8 × 10−2 M aqueous solution wasadded dropwise under stirring. Following the addition of citrate,the solution began to darken and turn bluish-gray or purple. Afterapproximately 10 min, the reaction was completed and the finalcolour of solution was a deep wine red. The solution was cooled toroom temperature under continuous stirring.

2.4. Synthesis of PCs heterostructured film

The ST–PEGMA 1100 colloidal dispersion was mixed with Aucolloidal solution (1:5 volume ratios). The hybrid film (ST–PEGMA1100–Au) was obtained by gravitational sedimentation of the col-loidal mixture and kept at 60 ◦C for 20 min.

2.5. Surface treatments of PCs heterostructured film

The hybrid film was previously immersed in G4 methanolsolution (10%) and kept for 24 h at room temperature. After thetreatment, the G4 modified film (ST–PEGMA 1100–Au–G4) wasdried at 30 ◦C for 6 h. In the next step, BSA solution was dropwiseadded on the surface of the film. The resulting film (ST–PEGMA1100–Au–G4–BSA) was kept at 24 ◦C for 24 h.

2.6. Characterization

The morphologies of polymer particles were investigatedthrough scanning electron microscopy (SEM) using a Philips XL-

30-ESEM TMP microscope. The samples were sputtered with athin layer of gold prior to imaging. The particles size measure-ment through dynamic light scattering (DLS) and the Z potentialwere obtained with a Nani ZS device (red badge). Transmission

290 A. Mocanu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 288– 293

agni

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3

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fi

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colloidal stability of the mixture. The size of the hybrid particlesbecomes 240 nm (Fig. 4). The monodispersity of the hybrid particleswas confirmed through the DLS data. Knowing the concentrationand the density for the polymer and Au colloids, respectively the

Fig. 1. Optical microscope images at various m

lectron microscopy (TEM) was performed on a Philips CM 120T microscope using an acceleration voltage of 100 kV. Atomicorce microscopy (AFM) was used for structural characterizationNT-MDTP47H microscope). All analyzed samples consisted inarticle films obtained by gravitational sedimentation. Micropho-ographs were recorded with an optical microscope (Olympus,X-41) equipped with a CCD camera. The UV/vis spectra wereecorded using a V-550 Jasco spectrophotometer. The fluorescencepectra were registered using a FP-6500 Jasco spectrofluorometerat 295 nm excitation wavelength).

. Results and discussion

The first stage in our study consisted in the preliminary char-cterization of the ST–PEGMA 1100 film using optical microscopy.he average distance between the defaults (resulted from water

vaporation) was estimated to 100 �m proving the good quality ofhe final opal films (Fig. 1).

In order to obtain more information about the ST–PEGMA 1100lm, respectively the particles size, SEM analysis was performed.

ig. 2. ST–PEGMA 1100 dispersion (a) SEM image and (b) AFM results of theT–PEGMA 1100 film.

fications; from left to right: 20×; 50×; 100×.

Fig. 2a is representative in this respect, showing a regular latticestructure, characteristic for PCs. The particle size of 200 nm is alsoconfirmed by AFM (Fig. 2b). The average roughness of the analyzedsurface was 27.3 nm.

PCs heterostructures [49] can exhibit a broader band gap bymanipulating the sphere sizes of the two constitutional com-ponents, which may offer functionality for engineered photonicbehaviour. With this aim, the ST–PEGMA 1100 colloidal dispersionwas mixed with Au colloidal solution. The size of Au parti-cles and the stability of colloidal mixture were investigated byTEM and DLS.

The diameter of metallic particles is around 10 nm (Fig. 3) andthe Z potential of the mixture is −45 mV. This value indicates the

Fig. 3. TEM image of Au nanoparticles.

Fig. 4. The DLS measurement for the colloidal mixture.

A. Mocanu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 288– 293 291

00×)

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w

Fig. 5. (a) Optical microscope images (20×; 50×; 1

olume of a single polymer and Au particles, it is easy to predict theumber of Au particles surrounding one polymer particle. Thus, 6u particles (3 positions for each 2 particles) have been distributed

round 1 polymer particle. The possible structure is inserted inig. 4.

The hybrid film obtained by mixing the two colloidal dispersionsas initially investigated using optical microscopy (Fig. 5a). Again,

Fig. 6. (a) Optical microscope image (20×; 50×; 100×) an

and (b) AFM results for ST–PEGMA 1100–Au film.

the distance between the defects (resulted from water evaporation)was around 100 �m, a proof of the good quality of the final opalfilm. The surface of the film was also analyzed by AFM, in order

to obtain information about the surface roughness (Fig. 5b). In thiscase the roughness of the surface increased from 27.3 to 32 nm,effect assigned to the generation of aggregates formed between Auand ST–PEGMA 1100 particles.

d (b) AFM results for ST–PEGMA 1100–Au–G4 film.

292 A. Mocanu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 392 (2011) 288– 293

00–Au–G4–BSA film (from left to right 20×; 50×; 100×).

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Fig. 8. UV–vis spectra for the films: ST–PEGMA 1100–Au–G4–BSA; ST–PEGMA1100; ST–PEGMA 1100–Au; ST–PEGMA 1100–Au–G4.

Fig. 7. Optical microscope image for ST–PEGMA 11

The next step consisted in the analysis of the ST–PEGMA100–Au–G4 hybrid film surface using AFM and opticalicroscopy. The G4 surface coating of the hybrid particles can be

asily noticed (Fig. 6). The roughness of the surface increased to9 nm, according to the G4 (4.5 nm) coating of Au nanoparticles.

In order to notice the influence of BSA on the surface ofT–PEGMA 1100–Au–G4, the film was treated with the protein.he first characterization consisted in macroscopic analysis of theurfaces using optical microscope (Fig. 7). The appearance of twoolours (green between the defects and red at the edge of defects)as noticed for the BSA-coated surfaces when compared to G4-

oated surfaces. The green colour may be due to the interactioncomplex) between BSA and G4 in the presence of the PCs het-rostructures. (For interpretation of the references to colour in thisext, the reader is referred to the web version of this article.)

In addition, UV–vis and fluorescence spectroscopy experimentsere performed in order to better explain the appearance of thelm colouration in the presence of BSA.

PCs present photonic band gaps – ranges of frequency in whichight cannot propagate through the structure. The enhancementf the intensity and the far stronger interaction of the connedight with any kind of material make PCs ideal candidates forptical sensing devices. To put in evidence the band gap in ourase, the UV–vis spectra were recorded. The characteristic bandap was identified at 560 nm for the ST–PEGMA 1100 film. In theases of ST–PEGMA 1100–Au and ST–PEGMA 1100–Au–G4–BSAlms, the band gap intensity decreased compared to ST–PEGMA100–Au–G4, where it disappeared. The decrease of the band gap

ntensity may be explained by the disturbance of the crystal struc-ure.

Unusual behaviour was noticed when comparing the signalsor ST–PEGMA 1100–Au–G4 (lack of reflection) and for ST–PEGMA100–Au–G4–BSA (reappearance of the band gap) (Fig. 8).

The fluorescence spectra of the ST–PEGMA 1100–Au–G4 filmre displayed in Fig. 9a with the aim of elucidating the unusualehaviour. The 556 nm emission intensity (for 295 nm excitationavelength) of the G4–BSA complex decreased when compared to4. Adding BSA did not modify the emission maximum wavelength.luorescence of tryptophan residues is very sensitive to the changesn their vicinity, thus it is widely used to study variations of the

olecular conformations of proteins. These observations supporthe hypothesis that surface groups and their charges are an impor-ant determinant of the dendrimer–protein interactions, typical of

simple collision quenching mechanism [21].Starting from this remark, the fluorescence spectra for

he studied films are presented comparatively in Fig. 9b.he fluorescence emission intensity decreased in therder ST–PEGMA 1100 > ST–PEGMA 1100–Au > ST–PEGMA100–Au–G4–BSA > ST–PEGMA 1100–Au–G4 > G4. The inten-

ity of the fluorescence emission for the films is similar withhe reflection behaviour in the UV–vis. The explanation could bettributed to the presence of PCs heterostructures. Fluorescence ofryptophan residues was amplified by the presence of the ordered

Fig. 9. Fluorescence spectra for the films: (a) G4-BSA; G4; (b) ST–PEGMA1100–Au–G4–BSA; G4; ST–PEGMA 1100; ST–PEGMA 1100–Au; ST–PEGMA1100–Au–G4.

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A. Mocanu et al. / Colloids and Surfaces A:

tructure. The type of interactions between the components willepresent the topic of future work.

. Conclusion

The present work presented the optical characterization of theultilayer film composed of ST–PEGMA 1100–Au–G4–BSA. The

ddition of the BSA-surface layer to the multilayer film led to annusual optical behaviour, namely the increase of the fluorescencemission intensity, and of the UV–vis reflection intensity, as com-ared to the ST–PEGMA 1100–Au–G4 film values. The explanationould be attributed to the presence of PCs heterostructures.

cknowledgments

The National Authority for Scientific Research from The Ministryf Education, Research and Youth of Romania is gratefully acknowl-dged for the financial support through the exploratory projectPolymeric Biomaterials For Bone Repair. Biomimetism Throughanostructured Surface”, PN-II-ID-2008-2, number 729/2009.

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