Article pubs.acs.org/JPCC
Amine Surface Modifications and Fluorescent Labeling of Thermally Stabilized Mesoporous Silicon Nanoparticles Wujun Xu,† Joakim Riikonen,† Tuomo Nissinen,† Mika Suvanto,‡ Kirsi Rilla,§ Bojie Li,∥ Qiang Wang,∥ Feng Deng,∥ and Vesa-Pekka Lehto*,† †
Department of Applied Physics, University of Eastern Finland, POB 1627, FI-70211 Kuopio, Finland Department of Chemistry, University of Eastern Finland, POB 111, FI-80101 Joensuu, Finland § School of Medicine, Institute of Biomedicine, University of Eastern Finland, POB 1627, FI-70211 Kuopio, Finland ∥ State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, P. R. China ‡
S Supporting Information *
ABSTRACT: Mesoporous silicon (PSi) has been shown to have extensive application opportunities in biomedicine, whereas it has frequently failed to produce complex systems based on PSi due to the lack of surface functional groups or the instability of the unmodified PSi surface. In the present study, PSi nanoparticles, stabilized by thermal oxidation or thermal carbonization, were successfully modified by grafting aminosilanes on the surface. The modifications were performed by covalently bonding 3-triethoxysilylpropylamine (APTES) or 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS) on thermally oxidized PSi (TOPSi) and thermally carbonized PSi (TCPSi). These materials were systematically characterized with N2 ad/ desorption, TEM, contact angle, zeta potential, FT-IR, 29Si CP/MAS NMR, and elemental analysis. To evaluate their application potentials, a fluorescent dye, fluorescein 5-isothiocyanate (FITC), was coupled on the surface of aminemodified nanoparticles. The effects of PSi matrix and surface amino groups on FITC coupling efficiency, fluorescent intensity, and the stability of fluorescence in simulated body fluid (SBF) were investigated. The nanoparticles modified with AEAPMS had higher FITC coupling efficiency than those modified with APTES. FITC-coupled TOPSi nanoparticles also possessed brighter fluorescence and better fluorescent stability in SBF. Furthermore, due to the protection caused by the mesoporous structure of PSi nanoparticles, the FITC-coupled TOPSi nanoparticles showed superior photostability in photobleaching experiment. diverse biofunctional molecules.6−9 Among these applications, utilization of amino groups to couple fluorescent agent is one of the most attractive topics in diagnostics and cellular imaging.8,9 Up to now, some efforts have been made to prepare fluorescent dye labeled silicon particles for bioimaging.10,11 However, in the previous reports, the fluorescent dye was either grafted on unmodified PSi materials or physically adsorbed in the mesopores of PSi. Moreover, no reports have been published about the long-term hydrolytic stability of fluorescence in biological fluids and the photostability of fluorescent silicon particles, which are of crucial importance for their applications in bioimaging. Herein, we report amine surface modifications of thermally stabilized PSi nanoparticles and their application in fluorescence labeling. The illustration of amine surface modifications of PSi is presented in Scheme 1. In order to stabilize the surface
1. INTRODUCTION Mesoporous silicon (PSi) possesses several attractive properties such as high surface area, large pore volume, controllable pore size distribution, and good biocompatibility.1 These advantages make PSi an attractive biomaterial for various biomedical applications.1−3 However, one problem frequently encountered when developing new applications of PSi nanoparticles is the instability of the unmodified PSi nanoparticles in biological environments. This consideration hinders the application of PSi, e.g., in long-term drug delivery. In our previous studies,2,4,5 it had been proved that thermal oxidization and thermal carbonization could not only improve the surface stability of PSi nanoparticles but also bring some interesting surface characteristics for drug delivery. Unfortunately, the lack of surface functional groups is still a problem for preparing novel complex drug delivery systems based on thermally stabilized PSi. Amine surface modification by aminosilane has been studied extensively in the field of biomedicine because amino groups have positive charge and active chemical property for coupling © 2012 American Chemical Society
Received: April 4, 2012 Revised: September 20, 2012 Published: September 22, 2012 22307
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Scheme 1. Illustration of Amine Surface Modifications of Thermally Stabilized PSi (a) and Chemical Structure of Aminosilanes, APTES and AEAPMS (b)
the carbonization, the PSi nanoparticles were treated with HF/ EtOH solution to remove the oxidized layer that had formed during the high-speed milling. The detailed preparation procedures of TOPSi and TCPSi are described elsewhere.4,10 2.3. Amine Surface Modifications of Thermally Stabilized PSi. Si−OH groups were utilized as active sites for amine surface modification. Therefore, in order to increase the surface density of Si−OH groups, the residual Si−H groups on TOPSi were further oxidized by NH3·H2O/H2O2/H2O (1/ 1/5, vol/vol) and HCl/H2O2/H2O (1/1/6, vol/vol) prior to the amine modification.12 The sample was rinsed three times in deionized (DI) H2O with sonication. Subsequently, the sample was dried at 65 °C overnight in air and at 85 °C for 2 h under vacuum. The oxidized TOPSi with the −OH groups was designated as TOPSi−OH. The amino modification was done by suspending 50 mg of TOPSi−OH nanoparticles in the mixture of aminosilane (APTES or AEAPMS) and anhydrous toluene. The mass concentration of aminosilanes was controlled at 2.0%. After reflux for 4 h, the PSi particles were recovered by centrifugation with the speed of 18 000 rpm. The residual aminosilanes were removed by rinsing the samples in ethanol three times with sonication. The obtained samples were dried at 65 °C for 2 h. The samples modified with APTES and AEAPMS were designated as TOPSi−NH2 and TOPSi−NH2D. The letter “D” represents the diamine (primary amine (−NH2) at the top and secondary amine (−NH−) in the middle of alkyl chain) of AEAPMS (Scheme 1). To produce active Si−H groups, TCPSi was directly treated with the solution of HF/EtOH.13,14 The subsequent surface oxidation with H2O2 and amine modifications of TCPSi were similar to those described for TOPSi. The TCPSi nanoparticles after surface oxidation with H2O2 and amine modification with APTES and AEAPMS were designated as TCPSi−OH, TCPSi−NH2, and TCPSi−NH2-D, respectively.
of PSi, the nanoparticles were thermally oxidized or thermally carbonized prior to amine modifications. Two kinds of commercially available aminosilanes, 3-triethoxysilylpropylamine (APTES) and 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS), were separately modified on the surface of thermally oxidized PSi (TOPSi) and thermally carbonized PSi (TCPSi) nanoparticles. Subsequently, fluorescein 5-isothiocyanate (FITC) molecules were coupled on the surface of PSi nanoparticles through the reaction between −NCS groups and surface −NH2 groups. There were two reasons for coupling FITC on the PSi nanoparticles. First, FITC molecules were utilized to evaluate the accessibility of surface amino groups, which is not only essential for further functionalization but also for potential applications such as grafting or adsorption of drug molecules. Second, the FITC fluorescent labeling itself is one of the representative applications of amino groups. The effects of PSi matrix and surface amino groups on the FITC coupling efficiency, fluorescent intensity, and the stability of fluorescence in simulated body fluid (SBF) were studied. In addition, as an important parameter for fluorescent particles, the photostability of FITC-coupled on PSi was also investigated.
2. EXPERIMENTAL SECTION 2.1. Preparation of Unmodified PSi Nanoparticles. A silicon wafer (100) of p+-type with the resistivity of 0.01−0.02 Ω cm was anodized in a HF/EtOH mixture (1/1; 38% HF). The detailed procedure to prepare free-standing PSi films for nanoparticles can be found in the study of Bimbo et al.10 After drying at 65 °C for 1 h, the obtained PSi films were ball-milled in ethanol. PSi nanoparticles were separated by centrifugation. 2.2. Thermal Stabilization of PSi Nanoparticles. PSi nanoparticles were oxidized at 300 °C for 2 h in air to produce TOPSi. TCPSi nanoparticles were prepared by a two-step thermal carbonization under a N2/acetylene (1/1) flow. Before 22308
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Figure 1. Nitrogen ad/desorption isotherm (a,d), pore size (b,e), and particle diameter distribution (c,f) of initial TOPSi (a,b,c) and TCPSi (d,e,f).
The images of the water drop on the film substrate were captured by a digital camera (Canon). The amine-modified PSi samples were also characterized with FT-IR, elemental analysis, and solid-state 29Si crosspolarization magic angle spinning nuclear magnetic resonance (29Si CP/MAS NMR) to detect the organic functional groups. The FT-IR analysis was carried out with Thermo Scientific Nicolet 8700. The amine content of the amine-modified nanoparticles was calculated from the results of CHN elemental analysis (CHNS analyzer, Vario MICRO cube). All solid-state NMR spectroscopy experiments were carried out at 7 T on a Varian Infinityplus-300 spectrometer, equipped with a Chemagnetic triple-resonance 4 mm probe, with resonance frequencies of 299.78 and 59.55 MHz for 1H and 29Si, respectively. The 29Si CP/MAS NMR spectra were acquired with a contact time of 2.0 ms, a repetition time of 1.0 s and a spinning speed of 12 kHz. The 29Si chemical shifts from PSi samples were referenced to tetramethylsilane (TMS). The fluorescence of samples was measured with Zeiss LSM 700 confocal laser scanning microscope (Zeiss, Germany). A 40× oil-immersion objective was applied for the fluorescence measurements. The fluorescent samples were excited with a 0.5 mW laser of 488 nm. The fluorescence intensity of samples was calculated from the microscopic images by the software of Zen 2009. The amount of FITC in solution was quantified with UV−vis spectrometer (JASCO V-530). 2.6. Fluorescence Stability of FITC-Coupled Nanoparticles. (1) Stability in SBF. FITC-coupled nanoparticles with the concentration of 1.0 mg/mL were dispersed in SBF with pH of 7.4. The samples were incubated at 37 °C under dark. After predetermined time intervals, 0.5 mL of solution with the suspended nanoparticles was sampled. The supernatant after centrifugation was collected to analyze the detached amount of FITC with UV−vis spectrometer at the absorption wavelength of 493 nm. To avoid the effect of FITC
Also, the TOPSi and TCPSi film samples were prepared to measure the samples with FT-IR and contact angle for convenience. The surface modifications of the film samples were the same as those for the nanoparticles. 2.4. Fluorescein Coupling. FITC (0.1 mg) was dissolved in 1.0 mL of absolute ethanol, and then, 1.0 mg of aminemodified nanoparticles were added to the solution. The reactants were stirred for 16 h at room temperature in a dark environment, 15 and solid products were recovered by centrifugation. The supernatant after centrifugation was collected to analyze the residue amount of FITC with UV− vis spectrometer at the characteristic absorption wavelength of 280 nm. The supernatant was diluted 1:15 with ethanol before actual measurement. The obtained nanoparticles were rinsed three times with ethanol and twice with DI H2O to remove the physically adsorbed FITC molecules. Three minutes of sonication was applied for each cycle. The amount of coupled FITC was quantified according to the Beer−Lambert law. The extinction coefficient (ε) of FITC in ethanol was 30 100 M−1·cm−1, which was calculated based on its concentration standard curve. 2.5. Material Characterization. The samples were characterized by N2 ad/desorption (Tristar II 3020, Micromeritics) to determine porous parameters. The specific surface area was calculated using the multiple-point Brunauer− Emmett−Teller (BET) method. The pore size distribution was calculated from the desorption branch using the Barrett− Joyner−Halenda (BJH) theory. High-resolution TEM (HRTEM) images were collected with JEOL JEM2100F. The zeta potential and the hydrodynamic diameter of the nanoparticles were measured with Malvern Zetasizer Nano ZS. The Huckel approximation with F(Ka) value of 1.00 was selected for the zeta potential measurement. Water contact angle was measured by placing a drop of DI H2O on the surface of the dried film. 22309
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Table 1. Pore Parameters, Contact Angle, and −NH2 and FITC Content of the PSi Samples sample
surface area (m2/g)
pore sizea (nm)
pore volume (cm3/g)
contact angle (deg)
TOPSi TOPSi−NH2 TOPSi−NH2-D TCPSi TCPSi−NH2 TCPSi−NH2-D
206.9 108.7 137.3 221.6 119.1 138.0
18.0 16.8 17.2 11.3 10.1 10.4
0.90 0.54 0.67 1.00 0.57 0.65
7.2b 26.2 37.2 15.0b 37.4 65.4
−NH2 contentc (mmol/g)
−NH2 density (1/nm2)
amount of coupled FITC (mmol/g)
0.91 0.57
2.71 1.70
0.18 0.19
1.30 0.63
3.53 1.71
0.13 0.15
a
The peak values of the pore size distribution. bTOPSi−OH and TCPSi−OH were used as the control samples for contact angle. cRefers to the primary amino groups.
The pore parameters and contact angles of PSi before and after amine modifications are summarized in Table 1. The decrease in the surface areas and pore volumes due to the surface modifications indicated that organic amines had been attached onto the surface of the PSi nanoparticles. The surface hydrophilic/hydrophobic properties of the PSi nanoparticles were characterized by contact angle measurements. The TCPSi−OH and TOPSi−OH possessed a typical hydrophilic surface due to the surface Si−OH groups. The introduction of alkyl groups from APTES or AEAPMS increased the contact angle, but their surfaces were still hydrophilic. This property is favorable for its potential applications in drug delivery.16 The HR-TEM images of PSi nanoparticles before and after surface modifications are shown in Figure S2, Supporting Information. According to the images, the pore diameters of TOPSi and TCPSi nanoparticles are in the mesopore range (5−40 nm). The calculated mean particle diameter and pore size are in agreement with the results shown in Figure 1 and Table 1. The zeta potentials versus pH of the nonfunctionalized and aminefunctionalized PSi nanoparticles are presented in Figure S3, Supporting Information. The isoelectric points (IEPs) of the nonfunctionalized TOPSi−OH and TCPSi−OH were 1.8 and 2.6. As expected, the amine modifications increased the surface charge of PSi nanoparticles. The IEPs of TOPSi−NH2, TOPSi−NH2-D, TCPSi−NH2, and TCPSi−NH2-D were 7.8, 8.8, 8.0, and 8.8, respectively. 3.2. Component Analysis of Amine-Modified PSi. Figures 2 and 3 show the FT-IR spectra of TOPSi and TCPSi before and after amine modifications, respectively. In the spectrum of the initial TOPSi (Figure 2a), the intense
protonation state on the extinction coefficient, the supernatant was diluted 1:5 with SBF before the actual measurement. The quantification of FITC detachment was based on the Beer− Lambert law in which the ε of FITC in SBF was 80 300 M−1·cm−1. The recovered nanoparticles were rinsed four times with EtOH and twice with HEPES buffer solution (10 mM, pH 7.4). Five minutes of sonication was applied in each cycle to remove the physically adsorbed or detached FITC molecules. Finally, the samples were measured with the confocal microscope in HEPES buffer solution to determine fluorescence intensity. (2) Photostability tests. Photobleaching experiments were also conducted with Zeiss LSM 700 microscope. FITC-coupled nanoparticles with concentration of 1.0 mg/mL were dispersed in HEPES buffer solution. A selected area was excited continuously with the 0.5 mW 488 nm laser. The intensity of the fluorescence was simultaneously recorded at 2.0 s intervals by the fluorescence microscope system. As the control sample, corresponding photobleaching test of pure FITC in HEPES was also carried out with the same procedure. The measurement of each sample was repeated three times with the same parameters. To present the results with standard error marks more illustratively, the results of fluorescent intensity after bleaching for 30, 60, 120, 180, and 240 s were adopted.
3. RESULTS AND DISCUSSION 3.1. General Properties of Thermally Stabilized PSi before and after Amine Modifications. The N2 ad/ desorption isotherms, pore size, and particle diameter distributions of the initial TOPSi and TCPSi nanoparticles are shown in Figure 1. The samples of TOPSi and TCPSi have similar broad pore size distributions typical for aggregated porous nanoparticles. Their maximum peaks are located at 18.0 and 11.3 nm, respectively. Figure S1 (Supporting Information) presents the N2 ad/desorption isotherms and pore size distributions of TOPSi film/microparticle samples and TCPSi film sample. All of these samples showed IV-type isotherms and narrow pore size distributions, indicating that these materials have characteristic mesoporous structures. The preparation procedures of film/microparticles are identical with their nanoparticles except for an absent/shorter milling step. Therefore, the broad pore size distribution was evidently caused by the heavy aggregation of the particles during drying and thermal treatments. This caused wide interparticle pores to form, which influences the interpretation of N2 ad/desorption isotherms and pore size distribution. The particle diameter distributions of the nanoparticles are shown in Figure 1c,f. The average particle diameters of TOPSi and TCPSi were 163 and 170 nm, respectively.
Figure 2. FT-IR spectra of TOPSi (a), TOPSi−OH (b), TOPSi−NH2 (c), and TOPSi−NH2-D (d). 22310
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Figure 3. FT-IR spectra of TCPSi (a), TCPSi−OH (b), TCPSi−NH2 (c), and TCPSi−NH2-D (d).
bands at 2259 and 2208 cm−1 indicated that a significant number of backbond oxidized Si−H groups still existed on the surface of TOPSi, though it had been oxidized at 300 °C for 2 h in air, as previously observed.12 The two bands disappeared after the oxidation treatments with NH3·H2O/H2O2/H2O and HCl/H2O2/H2O. In the spectrum of TOPSi−OH, the broad band between 3200 and 3700 cm−1, belonging to the asymmetric stretching of O−H, proved that the TOPSi−OH possessed an −OH-terminated surface. The sharp shoulder at 3738 cm−1 of the broad band was assigned to the stretching of free silanol groups. The Si−OH groups and H2O molecules adsorbed on the surface also displayed a band at 1630 cm−1. In the spectrum of TOPSi−NH2, the typical bands of −NH2 at 3371, 3296, and 1596 cm−1 were assigned to the asymmetric −NH2 stretch, symmetric −NH2 stretch, and deformation of hydrogen bonded amino group, respectively.17 The bands of C−H stretch were also observed between 2869 and 2933 cm−1. Compared with the spectrum of TOPSi−NH2, the main difference of TOPSi−NH2-D was the presence of a new band at 1261 cm−1, which was assigned to the rocking vibration of Si− CH3.18 The spectra of the TCPSi samples before and after the surface modifications shown in Figure 3 are comparable with the spectra of the TOPSi samples shown in Figure 2. In the spectra of TCPSi−NH2 and TCPSi−NH2-D, the deformation of the hydrogen bonded amino group at 1596 cm−1 was overlapped by the band from H2O. However, the new bands in the FT-IR spectra after modifications proved clearly that amino groups had been grafted onto the surface of TCPSi series samples. Solid-state 29Si CP/MAS NMR was used to identify the different kinds of silicon species in the nanoparticles. The obtained spectra are presented in Figures 4 and 5. In the spectrum of TOPSi−OH, three resonances at −110, −101, and −92 ppm were assigned to Q4, Q3, and Q2 of silicon species [Qn = Si(OSi)n(OH)4−n, n = 2−4], respectively.16 After the amine modifications, new intense resonance peaks appeared in the NMR spectra of TOPSi−NH2 and TOPSi−NH2-D. In Figure 4b, the two peaks of T3 and T2 at −69 and −65 ppm revealed the presence of Si(OSi)3SiR and Si(OSi)2Si(OH)R [Tm = Si(OSi)m(OH)3−mR, m = 1−3], respectively.19 In Figure 4c of TOPSi−NH2-D, apart from the peaks corresponding to the Q-bands between −80 and −120 ppm, the new signal at
Figure 4. 29Si CP/MAS spectra of TOPSi−OH (a), TOPSi−NH2 (b), and TOPSi−NH2-D (c).
−21 ppm was attributed to D2 silicon [D2 = Si(OSi)2R1R2], which was introduced from AEAPMS. Thermal carbonization with acetylene is an effective method to stabilize silicon-based materials. According to the computer simulation results of the previous studies,20,21 hydrogen leaves the silicon surface, and C atoms remain at temperatures higher than 670 °C. With the increase of temperature, C atoms can permeate into the framework of silicon to break Si−Si bonds and then Si1−yCy (0 < y
