Article pubs.acs.org/Langmuir
Amine Modification of Thermally Carbonized Porous Silicon with Silane Coupling Chemistry Ermei Mak̈ ila,̈ † Luis M. Bimbo,§ Martti Kaasalainen,† Barbara Herranz,§ Anu J. Airaksinen,∥ Markku Heinonen,‡ Edwin Kukk,‡ Jouni Hirvonen,§ Hélder A. Santos,§ and Jarno Salonen*,† †
Laboratory of Industrial Physics and ‡Materials Physics Laboratory, Department of Physics and Astronomy, University of Turku, Turku FI-20014, Finland § Division of Pharmaceutical Technology, Faculty of Pharmacy, and ∥Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki, Helsinki FI-00014, Finland S Supporting Information *
ABSTRACT: Thermally carbonized porous silicon (TCPSi) microparticles were chemically modified with organofunctional alkoxysilane molecules using a silanization process. Before the silane coupling, the TCPSi surface was activated by immersion in hydrofluoric acid (HF). Instead of regeneration of the silicon hydride species, the HF immersion of silicon carbide structure forms a silanol termination (Si−OH) on the surface required for silanization. Subsequent functionalization with 3aminopropyltriethoxysilane provides the surface with an amine (−NH2) termination, while the SiC-type layer significantly stabilizes the functionalized structure both mechanically and chemically. The presence of terminal amine groups was verified with FTIR, XPS, CHN analysis, and electrophoretic mobility measurements. The overall effects of the silanization to the morphological properties of the initial TCPSi were analyzed and they were found to be very limited, making the treatment effects highly predictable. The maximum obtained number of amine groups on the surface was calculated to be 1.6 groups/nm2, corresponding to 79% surface coverage. The availability of the amine groups for further biofunctionalization was confirmed by successful biotinylation. The isoelectric point (IEP) of amine-terminated TCPSi was measured to be at pH 7.7, as opposed to pH 2.6 for untreated TCPSi. The effects of the surface amine termination on the cell viability of Caco-2 and HT-29 cells and on the in vitro fenofibrate release profiles were also assessed. The results indicated that the surface modification did not alter the loading of the drug inside the pores and also retained the beneficial enhanced dissolution characteristics similar to TCPSi. Cellular viability studies also showed that the surface modification had only a limited effect on the biocompatibility of the PSi.
1. INTRODUCTION
surface area and provides a relatively stable, hydrophilic surface chemistry.17 Another method used for stabilization is the thermal carbonization of PSi with acetylene.18,19 This method provides almost complete surface coverage, resulting in a relatively nontoxic material20 suitable for gas sensing, drug delivery, and radiolabeling purposes21−23 with high chemical stability even in basic solutions. 17 The method may also be used in postfabrication pore size modification.24 Thermal carbonization is based on the adsorption of acetylene onto the surface of PSi and its subsequent absorption into the silicon structure under thermal treatment, providing a nonstoichiometric silicon carbide (SiC) layer.5 Utilizing the dissociation temperature of adsorbed acetylene and hydrogen desorption,25 the thermal carbonization process is divided into two distinct treatments, yielding at lower temperatures thermally hydrocarbonized PSi (THCPSi) and at higher temperatures thermally carbonized PSi (TCPSi).19,26
Mesoporous inorganic materials, such as porous silicon (PSi), have gained considerable attention due to their broad range of applications, ranging from different types of sensors, where the transducing may be electrical or optical,1−3 to optical coating of solar cells4 and biomedical applications, such as a drug carrier material5−9 and bioglasslike material.10,11 These applications are derived from the versatility of PSi, which allows easy control over its pore size, morphology, and porosity during the fabrication process and from the inherent high specific surface area that can be on the order of hundreds of m2/g. Essential for all the applications of PSi is the successful control of its surface chemistry, as the high specific surface area enhances the reactivity of the material. The Si−Hx covered surface of fresh PSi enables a number of different surface functionalization and stabilization treatments, but also necessitates them, as the hydrides are rather prone to oxidation under ambient conditions12 and are reactive toward various chemicals.13,14 A fairly common surface stabilization method for PSi is oxidation,15,16 which is usually done thermally in a gaseous phase. It enables an efficient coverage of most of the © 2012 American Chemical Society
Received: July 30, 2012 Revised: September 4, 2012 Published: September 11, 2012 14045
dx.doi.org/10.1021/la303091k | Langmuir 2012, 28, 14045−14054
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(Cemat Silicon S.A., Poland) with a resistivity of 0.01−0.02 Ω cm in a 1:1 (vol) aqueous HF (38%)−ethanol electrolyte with a current density of 50 mA/cm2 in the dark. The porous layer was detached from the substrate as a film by increasing the current density to the electropolishing region. The free-standing films were then milled with a high-energy ball mill (Pulverisette 7, Fritsch GmbH) in an agate grinding jar. The milled microparticles were wet sieved with ethanol in test sieves and size fractions of 1−25 μm and 53−75 μm were collected. 2.2. Thermal Carbonization of PSi. The PSi films and microparticles were initially stabilized by thermal carbonization with acetylene. The treatment method was a two-step carbonization at 500 and 820 °C, as described by Salonen et al.5 Briefly, the microparticles and films were first immersed into 1:1 (vol) aqueous HF (38%)− ethanol mixture for a few minutes in order to remove the native oxidation due the new surface formed in the milling or storage under ambient conditions, as the thermal carbonization process requires a fresh hydrogen-terminated PSi surface. After the subsequent drying, the PSi samples were inserted in a quartz tube under N2 flow (1 L/ min) for 30 min at room temperature to remove oxygen and adsorbed moisture. In the first step of carbonization treatment, an acetylene flow of 1 L/min was added for 15 min, after which the quartz tube was placed in a tube furnace at 500 °C for 15 min under 1:1 N2−acetylene flow. After the treatment, the tube was allowed to cool back to room temperature under N2 flow. For the second step, an acetylene flow was added for 10 min at room temperature followed by annealing of the sample for 10 min at 820 °C under N2 flow. Finally, the sample was allowed to cool back to room temperature under N2 flow. 2.3. Amino-Functionalization of TCPSi. The TCPSi samples were functionalized with 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) in order to covalently attach a −NH2 termination on the surface. Prior to the functionalization treatment, the TCPSi samples were immersed into 1:1 (vol) aqueous HF (38%)−ethanol solution for 5 min at room temperature in order to partially reactivate the stabilized TCPSi surface by producing hydroxyl groups for silanization.34 After the HF immersion, the samples were vacuum filtered from the solution and dried at 65 °C for several hours. The functionalization of free-standing TCPSi films and TCPSi microparticles was carried out in solutions consisting of different concentrations of APTES dissolved into anhydrous toluene (Merck KGaA). The HF-treated TCPSi was immersed into the APTES− toluene solution for 1 h at 25 °C. After the treatment, the APTES solution was removed and replaced with fresh anhydrous toluene. The samples were sonicated for 3 min in successive washing steps with fresh toluene, toluene−methanol, and methanol in order to remove loosely bound APTES from the porous structure. Finally, the samples were dried for 16 h at 65 °C. The functionalized samples are labeled as APSTCPSi (aminopropylsilane−TCPSi) with a suffix that indicates the concentration of the APTES solution used in the functionalization as volume percent. 2.4. Physical and Chemical Characterization. The porous properties of the different PSi samples were characterized with nitrogen sorption at 77 K with TriStar 3000 (Micromeritics Inc.). The specific surface area was calculated using the Brunauer−Emmett− Teller (BET) theory, and the pore volume was determined from the isotherm using the total adsorption value at relative pressure p/p0 = 0.97. The pore size distribution was calculated from the desorption branch of the isotherm using the Barrett−Joyner−Halenda (BJH) theory. The surface chemistry of the PSi samples was characterized using several supplementing analysis methods. Fourier-transform infrared (FTIR) spectrometric measurements were carried out with a Spectrum BX (PerkinElmer Co.) spectrometer in both transmission mode and with horizontal ATR accessory equipped with a diamond crystal (MIRacle ATR, Pike Technologies Ltd.). The measurements were taken between 4000 and 1000 cm−1, averaging 32 scans. X-ray photoelectron spectroscopic (XPS) measurements were performed with a PHI 5400 ESCA spectrometer (PerkinElmer Co.) with a Mg Kα X-ray source (hυ = 1253.6 eV). Survey spectra were collected with a pass energy of 89.4 eV and high-resolution multiplex spectra of the
Other methods used for stabilization and functionalization of PSi are hydrosilylation and silanization chemistry, which allow the grafting of various different chemical species onto the PSi surface. Lewis acid mediated27 or thermally activated28 hydrosilylation is often used in attaching the functionalizing molecule to the silicon surface. Silanization is also a suitable method for PSi functionalization. However, it is more commonly used with porous silica materials,29,30 as the silane coupling reactions usually proceed on oxide surfaces requiring a preoxidation of the PSi.6,31 Recently, Sciacca et al.32 demonstrated a method for coupling a dicarboxylic acid with a peroxide initiator onto THCPSi, yielding a partially COOH-terminated surface. Kovalainen et al.33 used another method for providing a carboxylic acid termination on THCPSi for peptide delivery by adapting the thermal hydrosilylation of undecylenic acid method introduced by Boukherroub et al.28 As different applications of PSi, such as drug delivery and biosensing, often require the occurrence of specific interactions between the porous host material and the guest molecules, the combination of the high surface coverage primary stabilization method with a secondary treatment that provides a specific functionalization offers a route to avoid possible limitations due to the incomplete coverage often associated with hydrosilylation and silanization.14 In this work, the approach was to enable the secondary functionalization of TCPSi, where the thermal carbonization would function as a chemical and structural stabilizer of the porous structure. The surface of TCPSi is natively covered with a thin oxide layer,23 which in part gives the material its hydrophilic nature. Though the native oxide layer could also be used as a starting point for further modifications, a functionalization process that takes advantage of the embedded SiC layer was pursued. In SiC structures, the dissolution of the surface oxides is terminated when hydrofluoric acid (HF) reaches the silicon oxide layer that is back-bonded to the carbon atoms.34 This treatment leaves the surface terminated with hydroxyl groups, instead of hydrides, as in the case of pure silicon structures, enabling the use of silanization for the secondary functionalization.34,35 3-Aminopropyltriethoxysilane (APTES) was selected as the organosilane for the surface modification due to its well-known properties29,36−38 and because it can provide the TCPSi a new secondary functionalization with amine group termination (−NH2). The utilization of amine-termination is common in immobilization of peptides and specific cells,39−41 making the NH2-covered surface very beneficial for biosensing purposes. In drug delivery, a positively charged surface enables the adhesion of the particles onto cell walls,42 where they may more effectively release the drug payload. The successfulness of the silane coupling is assessed by extensive characterizations of the new surface modification. The availability of the surface amine groups for further functionalization is demonstrated with biotinylation. The stability of the modification under physiologically relevant conditions is also discussed and the in vitro cytotoxicity of the microparticles is evaluated with Caco-2 and HT-29 cells. The applicability of material after functionalization is demonstrated with drug release studies of fenofibrate.
2. EXPERIMENTAL SECTION 2.1. Fabrication of PSi. The PSi was produced by electrochemically anodizing monocrystalline boron-doped p+-type Si(100) wafers 14046
dx.doi.org/10.1021/la303091k | Langmuir 2012, 28, 14045−14054
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Scheme 1. Schematic Representation of the Thermal Carbonization and Amino Functionalization of PSi with 3Aminopropyltriethoxysilanea
a
Layer thicknesses are not to scale.
main elements with a pass energy of 35.75 eV. All binding energies were referenced to the Si0(2p) peak at 99.3 eV. Linear background subtraction was performed for the measurement data before the peak fitting process. The background pressure during the measurements was
