gold hybrid bio-nanofilms prepared by layer-by-layer method

Colloids and Surfaces B: Biointerfaces 79 (2010) 276–283

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Optical and structural properties of protein/gold hybrid bio-nanofilms prepared by layer-by-layer method ˝ a , Andrea Majzik b , Imre Dékány a,b,∗ Edit Pál a , Viktória Hornok b , Dániel Sebok a b

Department of Physical Chemistry and Materials Sciences, University of Szeged, H-6720, Szeged, Aradi vt. 1., Hungary Supramolecular and Nanostructured Materials Research Group of The Hungarian Academy of Sciences, University of Szeged, H-6720, Szeged, Aradi vt. 1., Hungary

a r t i c l e

i n f o

Article history: Received 24 January 2010 Accepted 14 April 2010 Available online 21 April 2010 Keywords: Gold, Lysozyme Thin films Layer-by-layer QCM SPR

a b s t r a c t Lysozyme/gold thin layers were prepared by layer-by-layer (LbL) self-assembly method. The build-up of the films was followed by UV–vis-absorbance spectra, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) techniques. The structural property of films was examined by X-ray diffraction (XRD) measurements, while their morphology was studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM). It was found that gold nanoparticles (NPs) had cubic crystalline structure, the primary particles form aggregates in the thin layer due to the presence of lysozyme molecules. The UV–vis measurements prove change in particle size while the colour of the film changes from wine-red to blue. The layer thickness of films was determined using the above methods and the loose, porous structure of the films explains the difference in the results. The vapour adsorption property of hybrid layers was also studied by QCM using different saturated vapours and ammonia gas. The lysozyme/Au films were most sensitive for ammonia gas among the tested gases/vapours due to the strongest interaction between the functional groups of the protein. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Gold nanoparticles in the colloid size range enjoy popularity as a consequence of their widespread use in catalysis [1,2], photoelectron [3], biosensors [4,5], biomedicine and nanotechnology [6]. Nanoparticles in variable size and shape with nearly monodisperse dimensions can be produced by colloid chemical synthesis methods. The first replicable method has been developed by Turkevich, who prepared gold nanoparticles from gold-chloride with Na-citrate as a reductive agent [7]. Saraiva and Oliveira have pointed the fact that the concentration of reactants, the addition rate of citrate and the reaction temperature have significant effects on the size of forming gold nanoparticles [8]. He et al. have prepared sols with various particle size by changing the gold:citrate ratio. They have demonstrated that the colour of the sol changes with the particle size [9]. Majzik et al. have prepared gold NPs and nanorods that were stabilized and reduced by citrate reproducible in aqueous dispersions and on functionalized glass surface, respectively [10]. The formation kinetics of gold nanoparticles and nanorods have been studied and also the influence of modifications with cysteine and glutathione on the surface of Au nanoparticles.

∗ Corresponding author at: Department of Physical Chemistry and Materials Sciences, University of Szeged, H-6720, Szeged, Aradi vt. 1., Hungary. E-mail address: [email protected] (I. Dékány). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.04.010

Gold ion reduction can be replaced by the application of polyelectrolyte. Gold containing sols with a particle diameter of 12 nm were prepared in the presence of Poly(diallyl-dimethyl-ammoniumchloride) (PDDA) [11], and 25 nm particles in poly(ethylene-imine) (PEI) [12]. Furthermore, stabile sols can be prepared by acids like ascorbic acid [13] and 3-thiophenemalonic acid [14]. Various methods can be found in the literature for the preparation of thin films like magnetron sputtering [15,16], chemical vapour deposition [17], spin-coating [18] and Langmuir–Blodgett (LB) [19–24] technique. The layer-by-layer (LbL) method is based on self-assembly, that is perfectly applicable for the preparation of thin films. The LbL method is based on taking advantage of attractive interaction where on some sort of coating components of A and B with opposite charges are alternatively applied. During one deposition cycle, a film is immersed into component A. After the excess is removed by rinsing the thin film, the electrically charged components of A stay on the film making it possible for the oppositely charged component to bind onto the surface of the film. An (A/B)n hybrid film structure is obtained by repeating this action, where n is the number of deposition cycles [25]. Thin films of several composition can be generated by the above mentioned technique, e.g. polyelectrolyte/clay [26–28], polyelectrolyte/polyelectrolyte [29–30], polyelectrolyte/semiconductive nanoparticle [31–34], polyelectrolyte/layered double hydroxide [35,36], clay/peptide [37] and semiconductor/silica [38] layers, etc. Few publications specialize on the characterization of thin films

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from gold nanoparticles by LbL technique. There are some examples however for the preparation and characterization of films composed of gold/zinc sulphide [39], gold/polyelectrolyte [40,41] and gold/biomolecule [42,43]. The creation of thin films from gold nanoparticles and lysozyme by LbL method and interaction of gold nanoparticles and lysozyme has been studied in this presentation. The study has been carried out in a quasi 2D system by applying light absorption, QCM and SPR techniques and furthermore the structure (XRD) and morphological properties (SEM, AFM) of thin films have been determined. 2. Materials and methods 2.1. Preparation of citrate stabilized gold sol Au nanoparticles were prepared by Turkevich method [7]. The mixture of 0.4 cm3 of HAuCl4 solution (c = 0.05 mol/dm3 , HAuCl4 ·3H2 O, Sigma) and 13.85 cm3 of MQ water was heated until it boils under vigorous stirring, 5.75 cm3 of sodium citrate dihydrate solution (c = 0.034 mol/dm3 , C6 H5 O7 Na3 ·2H2 O, Aldrich) was added and kept stirring for the next 30 min. The colour of the solution would change until setting on wine-red. The sodium citrate acts as a reducing and stabilizing agent. The concentration of the gold sol is 0.02 g/100 cm3 while its pH is 6.2 and the zeta-potential of the gold nanoparticles is −27.32 mV. 2.2. Preparation of lysozyme/gold nanofilms Lysozyme/gold hybrid layers were prepared by layer-by-layer self-assembly method from the aquatic solution of lysozyme (pH 6.2, c = 0.001 g/100 cm3 , from chicken egg white, Sigma) and gold sol (pH 6.2, c = 0.02 g/100 cm3 ) on the surface of glass substrates (Menzel Superfrost microscope slides) and on QCM chrome/gold electrode surface using 5 MHz crystals. A previously cleaned (immersion in piranha solution for 3 min) substrate was first immersed into the lysozyme solution for 10 min followed by rinsing with distilled water and dried with nitrogen flow to obtain a lysozyme layer. Then the substrate was dipped into the gold sol

Fig. 2. (a) TEM image of the gold nanoparticles. (b) Size distribution of the gold particles determined from the TEM image.

for 10 min, after which it was again rinsed and dried. These steps were repeated n times – where n is the number of the deposition cycles, i.e. bilayer number –, to obtain (lysozyme/gold)n hybrid nanostructures (n = 10, 20, 30). 2.3. Methods The UV–vis absorption spectra of the gold sol and the nanolayers were measured by a Micropack Nanocalc spectrophotometer,  = 350–850 nm range. Quartz crystal microbalance (QCM) measurements were carried out by a Stanford Research System QCM 200 quartz crystal microbalance on a chrome/gold electrode (5 MHz) in a special measuring cell, under nitrogen atmosphere at 25.0 ◦ C. The SPR measurements were used for the investiga-

Fig. 1. UV–vis-absorbance spectrum of the gold particles.

Fig. 3. Zeta-potential of lysozyme solution at various pH.

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E. Pál et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 276–283 Table 1 The surface charge of gold nanoparticles and the lysosime at pH 6.2. Material

Charge (meq/g)

Gold NPs Lysozyme

−15.34 +83.97

tion of the build-up of the films on gold coated substrates in a flow cell at solid/liquid interface (SPRgoldTM substrate, Gentel Biosciences). The adsorbed amount was determined using the Sauerbrey equation:F = −Cf m,where F is the observed frequency change in Hz, Cf is the sensitivity factor for the crystal (56.6 Hz/mg−1 cm2 at room temperature) and m is the change in mass per unit area in g/cm2 . The electrokinetic (zeta) potential measurements were carried out in a Malvern Zetasizer Nano Series, Nano-ZS apparatus using a DTS 1060 folded capillary cells. For determination of the charge of particles Mütek PCD02 particle charge detector was used as streaming potential measurements. To determine the crystalline structure of Au NPs X-ray diffraction (XRD) measurements were carried out on a Bruker D8 Advance (Cu K␣ radiation, 40 kV, 30 mA) diffractometer at ambient temperature in the 30–80◦ 2 range. The morphology of gold NPs were examined by a Philips CM 100 transmission electron microscopy (TEM), and nanohybrid biofilms were examined by a field emission scanning electron microscopy (SEM) using Hitachi S-4700 type scanning electron microscope applying 10 kV accelerating voltage. Atomic force microscopy (AFM) measurements were taken on a Digital Instruments Atomic Force Microscope Nanoscope III in tapping mode, with a scanner capability of 12.5 ␮m in x and y direction and 5 ␮m in z direction. Silicon cantilever (Veeco Nanoprobe Tips RTESP model, 125 ␮m length, 300 kHz) were used. The scanning rate was 1 Hz. 3. Results and discussion

Fig. 4. Charge titration of Au sol with 0.001 g/100 cm3 lysozyme solution.

stabilized by Na-citrate can be seen in Fig. 1. The appearance of absorption maxima at 530 nm, i.e. the plasmon resonance of gold indicates the presence of individual gold NPs. The presence of individual particles is also proved by TEM images (Fig. 2a). Sphereshaped gold NPs with monodisperse size distribution were formed during synthesis with average diameter of 11.27 ± 1.45 nm according to the size distribution data of TEM images (Fig. 2b). A protein, lysozyme was used as binding material during film preparation. The pH of lysozyme solution was adjusted to 6.2. At this pH value, the average diameter of lysozyme is 4.8 nm determined by light scattering. Lysozyme possesses positive charge at this pH the zetapotential is +0.7 mV (Fig. 3). 3.2. Control of film build-up The negative surface charge of gold NPs was determined by charge titration of gold sol with 0.01 g/100 cm3 concentration of positively charged +24 meq/g PEI solution (chargePEI ). The specific charge of Au NPs was calculated from the following equation:

3.1. Characterization of film consisting materials chargeAu = Prior to film preparation the knowledge of surface and optical properties of film consisting materials is essential. The absorbance spectrum of gold nanodispersion of 0.02 g/100 cm3 concentration

VPEI × 0.01 × cPEI × chargePEI , cAu × VAu × MAu

where c and V are characteristic of the concentration and volume of the sol or solution, in g/100 cm3 for PEI solution and mol/dm3 for

Fig. 5. (a) UV–vis absorption spectra of the nanofilms. (b) Absorbance at 530 nm vs. bilayer number.

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Fig. 6. (a) Image of the gold sol and the thin films at different bilayer number in transmitted light. (b) Image of the thin films at different bilayer number in incident light. (c) UV–vis spectra and TEM images of gold sol before and after aggregation.

the gold sol. The charge of gold NPs is determined from the equivalence point came about to 15.34 meq/g Au (Table 1). The charge of lysozyme chains was also determined through titration with gold sol (that offered 83.97 meq/g lysozyme). This means that the charge of lysozyme chains is nearly six-fold relative to that of gold particles. The amount of protein relative to the amount of gold can be determined from the equivalency point of titration (Fig. 4) that resulted 15 mg lysozyme/g gold NPs (0.65 mg lysozyme/m2 gold NPs). The build-up of films was first followed by optical methods. The absorption spectra of films of different layer numbers are presented in Fig. 5a. The light absorption of films increases with layer number furthermore the shift from 620 nm to 654 nm of absorption maxima of films can be also observed. The interparticle interaction of Au NPs could be deduced during film formation from the disappearance of plasmon resonance maxima (530 nm) characteristic of individual Au NPs. Absorbance values at the maxima of the films in the function of layer number are presented in Fig. 5b. It can be seen, that the film build-up is close to linear, that is during build-up the adsorbed mass of single bilayers are nearly the same. To determine the film thickness, the deposited amount was calculated from the absorbance values by using a calibration curve

(absorbance vs. Au concentration). From the film coverage, which equals with the sol concentration in mg/cm2 , and an assumed density (at 1:1 lys/Au ratio  = 10.13 g/cm3 ) the film thicknesses can be determined, where AS is the surface coverage and  is the density of the film: t=

AS 

The thickness (t) of the film was determined from the UV–vis spectrum this way (Table 2). The photograph taken of gold nanodispersion and films of different layer number are presented in Figs. 6. It can be observed, that the colour of sol from the original reddish characteristic of individual particles changes to blue in thin film

Table 2 The particle size determined from the XRD patterns, thicknesses and porosity of the LbL films. n

d (nm)

tA 400 nm (nm)

tQCM (nm)

tSEM (nm)

tAFM (nm)

ε

10 20 30

11.3 11.2 10.4

33 74 118

17 43 75

430 ± 30 730 ± 90 1120 ± 140

32 74 126

0.47 0.36 0.40

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Fig. 7. Build-up of the film followed by QCM technique.

which also proves the aggregation of particles (Fig. 6a). The darkening of blue colour of films with layer number can be noticed as well (Fig. 6a) as the gold colour of films with different layer numbers in incident light (Fig. 6b). The explanation of change in sol colour from red to blue is that gold particles aggregate as a result of elution of some amount of lysozyme from the layer to the sol during immersion to gold sol. The TEM measurements and UV–vis spectra prove the particle aggregation (Fig. 6c). The shift of plasmon maximum from 523 nm to 620 nm with a second peak at 765 nm is observed in the UV–vis spectra of the sols. Two plasmon peaks are generally characteristic of nanorods or rod-like appearance of spherical particle aggregate. The build-up of films can also be followed by QCM technique. The results of QCM experiments also show the linear build of films (Fig. 7). The average mass increase of the film for one lysozyme/Au bilayer is 2.33 ␮g/cm2 according to the slope of the QCM film formation curve.

Fig. 9. (a) Schematic illustration of SPR measuring system at S/L interface. (b) Shift of the plasmon of the gold substrate during the film deposition. (c) Change of the angle with the bilayer number.

Fig. 8. XRD patterns of the lysozyme/gold films at different bilayer number.

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Fig. 10. AFM image (3 ␮m × 3 ␮m) and section analysis of the surface of the lysozyme/gold film (n = 30).

The X-ray diffractograms of films with different layer number can be seen in Fig. 8. The reflections characteristic of cubic gold appear in the diffractograms besides the increase of reflections with layer number can be as well observed. The size of layer constructing particles the can be determined from the diffractogram by means of the Scherrer-equation (Table 2). The size of particles show good agreement with that of determined from TEM investigations as it is evident from the values of Table 2. The authors can make the conclusion that during layer build aggregates form, which contain the individual particles that are able to communicate each other thus the plasmon resonance of individual particles shifts. The setup of measuring apparatus at S/L interface and the control of layer build-up by SPR method can be seen in Fig. 9a and b, respectively. It is important to notice that the degree shift is higher during gold nanoparticles adsorption than in case of lysozyme layer buildup. In Fig. 9c the change in degree shift is presented in the function of layer number. Both the QCM and SPR sense higher amounts of gold binding to the Au surface compared to the lysozyme layer as higher mass increase or  shift can be observed as the effect of gold. Approximately 19.2◦  shifts in plasmon experiments corresponds to 1 ␮g/cm2 adsorption on quartz crystal in case of lysozyme, while

this value is 1.9 for Au. Is it obvious that the layer build-up is about linear if the data points concern to the build-up of one particular type of materials are regarded. 3.3. Morphological characteristics of films The morphology of film surface was characterized by atomic force microscopy. A typical AFM image and cross-sectional analysis of film surface can be seen in Fig. 10. Following AFM investigations it is established that the surface of the film is relatively smooth divided by some nm high and several hundred nm hillocks on the surface. The roughness of the 3 ␮m × 3 ␮m area in the figure is 33.6 nm. The thicknesses of the films were determined by direct methods with the aid of cross-sectional SEM (Fig. 11a) and AFM images (Fig. 11b). The determined layer thickness values of films with different layer numbers are summarized in Table 2. If we compare the values determined from the absorbance spectra read at 400 nm wavelength to the results of QCM measurements, respectively, it can be established that there is good agreement between the layer thicknesses determined by various methods (AFM, A, QCM). Pores

Fig. 11. (a) Cross-section SEM image of the lysozyme/gold film (n = 30). (b) Thickness determination by AFM.

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Fig. 12. Schematic illustration of film structure (orange: gold nanoparticles, green: lysozyme chains). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

in the film structure explain the difference between the QCM and AFM data. The adsorbed mass of the films divided by the film density results the QCM thickness values. The presence of pores is not included in this calculation. Consequently the QCM thickness assumes compact layers, while the AFM measurements reveal the real, porous structure. Thus the porosity was determined by the following formula: ε=

Vt − VQCM tAFM − tQCM = Vt tAFM

where Vt is the total volume, tAFM is the thickness of the film based on AFM measurements calculated from the film thickness and film surface, and VQCM is the volume, tQCM is the thickness of the film determined from QCM experiments, respectively. These values are presented in Table 2. The SEM measurements resulted in order of magnitude difference. This deviation is partially due to the pores and the loose, weakly bonded structure of the films that were damaged along the breaking line which were formed during film breaking in order to determine their thickness. This broken structure can be seen in Fig. 11a. The assumed structure is exhibited schematically in Fig. 12.

Fig. 13. (a) Frequency of 30 layers lysozyme/gold film on QCM crystal measured in water, ethanol and toluene vapours. (b) Frequency of 30 layers lysozyme/gold film on QCM crystal measured in ammonia and acetic acid.

3.4. Vapour adsorption properties of films The adsorption properties of the 30 layers of film were studied by using QCM technique. During experiments the 30 layers of film built onto the surface of QCM crystal was placed into saturated atmosphere of polar and apolar vapours. The adsorption was reversible in every case, i.e. the adsorbate was removable from the films by nitrogen gas flow subsequent to the tests. During QCM measurements some period of time is necessary in nitrogen atmosphere to reach equilibrium (about 15 min in Fig. 13a) to obtain the frequency characteristic of the 30 layer film. Significant decrease in frequency is observable as the affect of vapour exposition and adsorption when some aliquot of water, ethanol or toluene is poured at the bottom of the measuring cell (Fig. 13a). The frequency jump is more apparent in case of ammonia relative to acetic acid vapours (Fig. 13b). The reason for this is that the acetic functional groups of the protein are electrostatically bound to the gold NPs to form alternating lysozyme/Au layers, while the alkaline groups of the amino acids remain free to attach ammonia gas. Thus ammonia gas could interact with the appropriate functional groups of the protein. The adsorbed mass by the film was plotted in Fig. 14 in cases of various vapours/gas. It can be noticed, that it could adsorb ammonia gas to the highest extent. The adsorbed amount of vapour relative to the monomolecular coverage can be determined in the knowledge of surface cross-sectional molar area (am ) [44,45] according to the following equation: =

m × am × 10, M

Fig. 14. Adsorbed amount of different vapours determined by QCM.

where m is the material amount of the adsorptive in ␮g/cm2 on the 30 layer lysozyme/Au nanofilm determined by QCM (Table 3). The am value is in m2 /mmol according to reference [44], M is the molar mass of adsorptive. Among the studied vapours only ammonia exceeds significantly the total coverage relative to one monolayer due to the chemical interaction with the carboxylic groups of the amino acids. The adsorption of polar vapours (water Table 3 Adsorbed amount of adsorptive on 30 layer Lys/Au nanofilm: surface loading: 76 ␮g/cm2 , 0.239 mg material on glass surface. Adsorbed molecule

am (m2 /mmol)

m/QCM (␮g/cm2 )



/#nmono

Water Acetic acid Ammonia Ethanol Toluene

102 174 78 150 240

1.41 3.00 10.1 2.22 1.90

79 87 463 73 50

1.32 1.45 7.72 1.22 0.82

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and ethanol) is favoured on hydrophilic surfaces, like in case of our bio-nanofilms, while the hydrophobic surfaces, e.g. surface modified layer silicates adsorbs aromatic components [46]. Therefore we examined also the adsorption of toluene, regarding that the proteins contain hydrophilic segments (e.g. ␤-turns) in order to examine their participation in the adsorption. For one layer hybrid film the hydrophilic/hydrophobic ratio is 1.62:1 based on the comparison of /#nmono data for hydrophilic (water, ethanol) and hydrophobic (toluene) molecules (Table 3). According to the vapour adsorption measurements it can be established that in case of suitable functionalization of gold nanoparticles the utility of lysozyme/gold films as vapour/gas sensors can be possible. 4. Conclusion Lysozyme/gold thin layers were prepared by layer-by-layer selfassembly method from gold NPs and aquatic lysozyme solution. The build-up of the films proved to be close to linear according to three independent methods like vis-absorbance, QCM and S/L interface SPR techniques. It was found that gold NPs had cubic crystalline structure, the primary particles form aggregates in the thin layer due to the presence of lysozyme molecules. The spectrophotometric measurements prove change in particle size while the colour of the film changes from wine-red to blue. The layer thickness of films was determined by four independent ways (Vis, QCM, SEM and AFM) and the film porosity explains the difference in the results. The adsorption property of hybrid layers was also studied by QCM using different saturated vapours and gas. The lysozyme/Au films were most sensitive for ammonia gas among the tested gas/vapours due to the electrostatic interaction between the free acidic groups of the protein, while the alkaline functional groups participate in film formation through lysozyme-Au interlayer interaction. Acknowledgement The authors are very thankful for the financial support of the Hungarian National Scientific Fund (OTKA) Nr. K73307 and NK 73672. References [1] T.F. Jaramillo, S.H. Baeck, B.R. Cuenya, E.W. McFarland, J. Am. Chem. Soc. 125 (2003) 7148. [2] J. Strunk, K. Kähler, X. Xia, M. Comotti, F. Schüth, T. Reinecke, M. Muhler, Appl. Catal. A 359 (2009) 121. [3] J.H. Park, Y.T. Lim, O.O. Park, J.W. Yu, Y.C. Kim, Chem. Mater. 16 (2004) 688. [4] G. Yang, R. Yuan, Y.Q. Chai, Colloid. Surf. B: Biointerf. 61 (2008) 93. [5] Z.X. Zhao, H.C. Wang, X. Qin, X.S. Wang, M.Q. Qiao, Colloid. Surf. B: Biointerf., doi:10.1016/j.colsurfb.2009.01.011.

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