Si nanocomposite layers

Materials Science and Engineering C 27 (2007) 1439 – 1443 www.elsevier.com/locate/msec

Gold and silver/Si nanocomposite layers Irina Kleps a,⁎, Mihai Danila a , Anca Angelescu a , Mihaela Miu a , Monica Simion a , Teodora Ignat a , Adina Bragaru a , Lucia Dumitru b,1 , Gabriela Teodosiu b,1 a

National Institute for Research and Development in Microtechnologies (IMT–Bucharest), P.O. Box 38-160, Bucharest, Romania b Institute of Biology, 296 Splaiul Independentei, P.O. Box 56-53, Bucharest, 060031, Romania Received 5 May 2006; received in revised form 15 June 2006; accepted 22 June 2006 Available online 8 August 2006

Abstract Ag and Au nanolayers were realised by physical and chemical deposition methods on porous silicon (PS) nanostructured surfaces for biomedical applications: support for living cells, biodegradable material for the slow release of drugs/minerals, and as a bioactive material for scaffolds. Au nanoparticles on nanocrystalline Si are widely used in increasing substrate biocompatibility properties. It has an electrochemical potential of + 0.332 mV and surface energy around 25 erg/cm2, close to those of living tissues. The Au nanocrystallites orientation on nanocrystalline Si substrates is also of great interest for application in biochemistry; the Au (111)/nc-Si surface has a higher density of atoms compared with Au (100); this favours the attachment of a higher number of atoms and bio-molecules on the gold surface. Ag nanoparticles on nanocrystalline Si are important for the latter's anti-microbial properties. In minute concentrations, Ag is highly toxic to germs while relatively non-toxic to human cells. Microbes are unlikely to develop a resistance against silver, as they do against conventional and highly targeted antibiotics. The Au and Ag nanoparticles/silicon nanocomposite layers as-deposited and thermally treated were investigated by optical microscopy, X-ray diffraction, and biological tests using eukaryotic and prokaryotic cell cultures. The experimental results sustain the use of Au/Si and Ag/Si or combined Ag/Au/Si nanocomposite structures as biocompatible and anti-microbial matrix. © 2006 Elsevier B.V. All rights reserved. Keywords: Nanocomposite layers; Gold; Silver; Porous silicon; X-ray diffraction; Biocompatibility

1. Introduction The interactions between the organic/biological materials and the metallic solid surfaces were intensively studied in recent years due to their multiple applications [1]. Advances in the field of nanotechnology have provided new forms of gold and silver available for use in biosensors or in implantable devices. In this paper gold and silver thin layers deposited on porous silicon (PS) substrates were investigated for various biological applications. The (111) gold layers are of great interest in self-assembled monolayers (SAMs) because they can be used as chemical linkers for the attachment of a new material layer, for protein, enzyme and oligonucleotide bio-immobilization, or in molecular electronics. ⁎ Corresponding author. Tel.: +40 21 4908085; fax: +40 21 4908238. E-mail addresses: [email protected] (I. Kleps), [email protected] (L. Dumitru), [email protected] (G. Teodosiu). URL: http://www.imt.ro (I. Kleps). 1 Tel.: +40 21 223 90 72; fax: +40 21 221 90 71. 0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.06.040

On the other hand, the nanocrystalline silver films in biological media activated to release silver clusters of AgO, silver cations and silver radicals are important for their antimicrobial properties associated with other healing effects [2]. Meanwhile, porous silicon (PS) known as nano-structured silicon, aside from its known luminescence properties, has recently attracted much attention because of its biocompatible, bioactive and biodegradable nature [3]. PS offers a surface topography controllable with nanometer resolution in three dimensions and allows chemical surface modifications. 2. Experimental data The PS layers, used as support in the experiments, for the physical vapour deposition of gold layer, were obtained on Si wafers: Si(100), p+ type, 0.005 Ω cm. The experimental conditions related to the sample preparation are presented in Table 1. Determined by gravimetric measurements, the porosities of PS layers used in these experiments are 38% and 60%. Gold and

1440

I. Kleps et al. / Materials Science and Engineering C 27 (2007) 1439–1443

Table 1 Experimental conditions Nanocrystalline Si (porous silicon)/Si p+ (100)

Porous silicon fabrication conditions

PVD-Au 100 nm

PVD-Ag 100 nm

Group 1 5 nm pore diameter – PS

– 25% HF concentration – Time: 30 min

Au-RX3 Au-RX1

Ag-RX4 Ag-RX5

Porosity 38%

– Anodic current density: 3 mA/cm2

Au-RX2

Ag-RX6

Group 2 15 nm pore diameter – PS Porosity 60% Si p+ (100) – reference sample

– 25% HF concentration – Time: 30 min – Anodic current density: 20 mA/cm2 –

Au-RX6 Au-RX4 Au-RX5 Au-RX8

silver layers of 100 nm thickness were deposited by thermal evaporation on the PS substrates. The Au/PS/Si and Ag/PS/Si nanocomposites layers were thermal treated in reducing atmosphere (H2 and N2). AgNO3 salt and AgNO3 1% ethanolic solution were also deposited on the PS/Si substrate in order to compare the bactericide effect of PVD-Ag and AgNO3 on the PS/Si substrate. The variation of the structural characteristics of gold and silver deposited on Si nanostructured layers as a function of temperature

AgNO3 (salt and 1% solution) Ag-RX2 (salt) Ag-RX1 (1% H2O solution)

Thermal treatment (°C) As-deposited 500 900 As-deposited 500 900 900

annealing (at 500 °C and 900 °C) were investigated by X-ray diffraction method, comparatively to as-deposited samples. 3. Results and discussion 3.1. X-ray diffraction characterization Two groups of Au/PS and Ag/PS were analyzed by XRD methods with the aim to establish the subsequent annealing influence (500 and 900 °C) on the deposited film microstructure on the porous substrate. XRD measurements were made using a DRON-3 diffractometer in θ–2θ angular scanning configuration and both the filtered continuous emission spectra (Co Kα1,2 – Ag films) and the double crystal method (monochromatic Mo Kα1 – Au films). The mean average size (Scherrer equation) of the gold crystallites calculated from X-ray diffraction patterns (Fig. 1) are presented in Table 2. XRD analyses of Au/PS samples belong to group 1 (38% porosity) (Fig. 1a) and reveal the following aspects: (i) in the as-deposited films, the initial nucleation begin on Au(220) planes; this process is rapidly blocked; a low value of the (220) diffraction peak intensity is observed; the crystallization process continues and the Au(111) peak becomes dominant; (ii) after thermal annealing at 500 °C, the crystallization process on the (111) planes continues, and a (111) texture is obtained, with (220) maximum nanocrystals size; (iii) thermal annealing at 900 °C induces an increase of the (111) crystallites, indicating a clear crystallization on (111) planes, leading to a (111) textured surface. Table 2 Average size of the gold crystallites (Dhkl)

Fig. 1. X-ray diffraction pattern of Au/PS for as-deposited and annealed samples, MoKα1: (a) group 1 and (b) group 2.

Sample

AuRX3

AuRX1

AuRX2

AuRX6

AuRX4

AuRX5

AuRX8

T (°C)

Asdeposited

500

900

Aseposited

500

900

900

Dhkl (nm)

Dhkl (nm)

Dhkl (nm)

Dhkl (nm)

Dhkl (nm)

Dhkl (nm)

Dhkl (nm)

18.08 18.30 39.02 12.77 6.26 20.36

19.79 16.97 23.17 15.20 19.72 19.63

28.26 25.64 – – 25.16 26.95

13.84 10.70 31.64 9.50 10.38 14.46

22.09 17.56 27.21 18.03 16.06 19.83

32.06 21.52 23.32 – – 22.16

27.10 24.89 – – 25.29 26.24

Peak 111 200 220 311 222 D (nm)

I. Kleps et al. / Materials Science and Engineering C 27 (2007) 1439–1443

1441

From the point of view of the microstructure analysis on the Ag films on PS, the annealing treatment at 500 °C has no effect (crystallization continue on (200) planes, and the films have a higher crystalline content), while annealing at 900 °C produces a mixed (111) and (200) texture, with bigger grain sizes (Fig. 2a and b). The Ag films deposited on a diluted solution of AgNO3 are in an initial stage of crystallization, with a high amorphous content, deduced from the large amorphous hump centred at 44.5° and from the high background and very low intensities of the corresponding Ag diffracting planes (Fig. 3). This fact can be explained by the low content of Ag atoms on the surface, and thus by the resulting lower thickness of the deposited Ag crystalline layer. Although the total thickness of the layer may be bigger than in the case of the samples Ag-RX4 – Ag-RX6 (the diffracted intensity from the (400) Si is lower), the crystalline content is very low (very low diffracted intensities from Ag atomic planes). The films deposited from salt are the thickest and with the lowest crystalline content. 3.2. Au(111)/PS/Si nanocomposite structures for biological applications

Fig. 2. X-ray diffraction pattern of Ag/PS samples after thermal annealing at 500 °C (a) and 900 °C (b) comparatively to as-deposited samples.

XRD analyses of samples belonging to group 2 (60% porosity) (Fig. 1b) reveal the following aspects: (i) in the as-deposited films, and in 500 °C thermally treated samples, the initial crystallization on (220) planes is stopped, and nucleation began on Au (111) planes more rapidly compared to group 1; (ii) related to the surface texture, the thermal annealing at 500 °C leads to: weak crystallinity (one order of magnitude lower) compared to the less porous layer, treated at 900 °C; stronger crystallization on (111) planes than in the group 1 samples, treated either at 500 °C or at 900 °C. All samples, as-deposited and annealed from group 2, present lower XRD intensities than those of group 1, on (111), (220) and (200) orientations. The crystallite size significantly increases with thermal annealing with 5–8 nm from 500 °C to 900 °C, reaching the same size, 25–28 nm, excluding the (220) and (311) crystallites which disappeared. For the samples of group 2, treated at 900 °C, the growth of crystallite dimensions reaches a maximum for (111) planes, simultaneously with the high decrease of (111) orientation (Table 2).

Nanocrystalline Au(111)/PS/Si structures were used and presented good results for the self-assembly of biological material or as biocompatible matrix, while Ag/PS/Si structures have antibactericide properties. Gold and silver /PS nanocomposite layers on the same biochip can confer it as interesting biological properties for many applications, either due to the metallic nanocrystallite film or to the porous silicon layer [4–6]. The Au nanocrystallites orientation on nanocrystalline Si substrates is of great interest for application in biochemistry; the Au(111)/nc-Si surface has a higher density of atoms compared with Au(100); this favours the attachment of a higher number of atoms and biomolecules on the gold surface. Nanocrystalline Au(111) films have important applications in biochemistry, especially in selfassembly of the thiol-end organic molecules (SAM), such as HS-

Fig. 3. X-ray diffraction pattern of AgNO3 (1% solution)/PS samples after thermal annealing at 500 °C.

1442

I. Kleps et al. / Materials Science and Engineering C 27 (2007) 1439–1443 Table 3 The growth evolution of bacterial cells from silver/porous silicon plates No.

1 2 3 4 5 5D

Fig. 4. Immobilisation of a fluorescent oligonucleotide on Au/Ps (40×, Nikon fluorescence microscope).

(CH2)6-OH, p-aminothiophenol, octadecanethiol, and DNAprimer molecule (HS-(CH2)6-5′-GGC-CAT-CGT-TGA-AGATGC-CTC-TGC-C-3′). The thiol-end DNA primer/Au/Si was used for a polymerase chain reaction (PCR) microchip. The best results as support membrane of a polymerase chain reaction (PCR) microreactor for the DNA fragments amplification were obtained using gold on PS of lower porosity, treated at 500 and 900 °C with a high (111) texture. Fig. 4 shows the immobilisation of a fluorescent oligonucleotide on the Au(111)/PS/Si microreactor membrane. On the other hand, Au/PS matrix presents good biocompatibility because it has an electrochemical potential of +0.332 mV and surface energy of 25 erg/cm2, close to those of living tissues. Au/PS was used as support for the hamster ovarian cells (CHO). It can be observed that the inorganic substrate (Au/PS/Si) is a biocompatible material, appropriate for cultivating adherent cells in vivo and without noticeable toxicity. It is also important to emphasize that no further coating with polylysine or collagen was required (Fig. 5). 3.3. Ag/PS/Si antibactericide properties In order to investigate the antibactericide properties of the Ag/ PS structures, a heterotrophic bacterium isolated from natural

Sample

Ag-RX2 Ag-RX6 Ag-RX5 Ag-RX1 Ag-RX4 PS

Cellular growth (OD = 660 nm) 24 h

48 h

72 h

0.862 0.885 0.711 0.931 0.696 1.474

0.931 1.088 0.924 1.186 0.693 1.462

0.981 1.336 0.904 1.142 0.831 1.464

aquatic habitat, characterized as Gram-positive, rod-shaped, endospore-forming bacteria, from Bacillus genus was used. A specific protocol was applied, as follows: Microorganisms were grown on a solid medium represented by nutrient agar slant. Cultures were incubated at 30 °C, for 24 h. After this period, the microorganisms in the exponential growth phase were inoculated in nutrient broth, in Erlenmeyer flasks. For each culture a preinoculum on liquid medium was performed, which was afterwards used at a 1/10 rate to initiated new cultures. Cultures were incubated at 30 °C, under continuous stirring (200 rpm), in the dark. Subcultures, at which plates of porous silicon were added, were performed from these cultures. The subcultures were incubated in static conditions, at 30 °C. The plates of Ag/PS/Si were taken over from the cultures and immersed in sterile conditions, in flasks with sterile culture media. These were incubated in static conditions, at 30 °C, for 72 h. The growth of microorganisms was monitored by spectrophotometric determination of cellular density (OD) at 660 nm, at 24-h interval, for 72 h, and the results are presented in Table 3. The maximal growth was recorded in the same sample (5D) represented by untreated porous silicon plates (1.464 U.D.O). The growth of strain used in experiments was reduced in the samples with silver (variants 1–5), where the cellular densities obtained were between 0.831 U.D.O. (Ag/PS, untreated) and 1.336 U.D.O. (Ag/PS, 900 °C). The results can be correlated with the silver nanocrystallite size in the samples treated at a higher temperature. It was observed that the antibactericide properties of silver are more pronounced in the Ag/PS samples which have smaller nanocrystallites (higher surface). 4. Conclusions

Fig. 5. CHO cells on samples with Au/PS/Si.

XRD analyses of the gold films deposited on porous silicon substrates and thermally treated at temperatures higher than 500 °C emphasize the (111) crystallite orientation. The crystallite size significantly increases with thermal annealing with 5–8 nm from 500 °C to 900 °C, reaching the same size, 25–28 nm, excluding the (220) and (311) crystallites which disappeared. In the case of Ag/PS samples, the annealing treatment at T = 500 °C has no effect from the point of view of the microstructure analysis of the Ag films on PS (crystallization continues on (200) planes, the films have a higher crystalline content), while annealing at 900 °C produces a mixed (111) and (200) texture, with bigger grain sizes.

I. Kleps et al. / Materials Science and Engineering C 27 (2007) 1439–1443

The nanocrystalline gold, (111) textured deposited on PS layers were successfully used in biomedical applications, such as PCR membrane functionalization, or as support for living cells. Antibactericide properties of the Ag/PS/Si structures were demonstrated in Gram-positive, rod-shaped, endospore-forming bacteria culture. References [1] H. Basch, M.A. Ratner, J. Chem. Phys. 120 (12) (2004) 5771. [2] F. Fu-Ren, A. Bard, J. Phys. Chem. 106 (2002) 279.

1443

[3] L. Canham, United State Patent, 6,322,895 (2001). [4] A. Angelescu, I. Kleps, M. Miu, M. Simion, T. Neghina, A. Bragaru, S. Petrescu, C. Paduraru, A. Raducanu, N. Moldovan, Rev. Adv. Mater. Sci. 5 (2003) 34. [5] W. Lang, P. Steiner, F. Koslowski, in: J.C. Vial, J. Derrien (Eds.), SpringerVerlag, Les Editions de Physique, (1994) 293. [6] I. Kleps, A. Angelescu, M. Miu, in: G.M. Chow, I.A. Ovid'ko, T. Tsakalakos (Eds.), NATO Science Series 3, vol. 78, 2000, p. 337.