Quantitative Analysis of Porous Silicon Nanoparticles Functionalization by 1H NMR

Porous silicon (PSi) nanoparticles have been applied in various fields, such as catalysis, imaging, and biomedical applications, because of their large specific surface area, easily modifiable surface chemistry, biocompatibility, and biodegradability. For biomedical applications, it is important to precisely control the surface modification of PSi-based materials and quantify the functionalization density, which determines the nanoparticle’s behavior in the biological system. Therefore, we propose here an optimized solution to quantify the functionalization groups on PSi, based on the nuclear magnetic resonance (NMR) method by combining the hydrolysis with standard 1H NMR experiments. We optimized the hydrolysis conditions to degrade the PSi, providing mobility to the molecules for NMR detection. The NMR parameters were also optimized by relaxation delay and the number of scans to provide reliable NMR spectra. With an internal standard, we quantitatively analyzed the surficial amine groups and their sequential modification of polyethylene glycol. Our investigation provides a reliable, fast, and straightforward method in quantitative analysis of the surficial modification characterization of PSi requiring a small amount of sample.


APSTCPSi Nanoparticles Fabrication
The nanoparticles were produced by following the fabrication steps outlined previously. [1][2][3] In detail, multilayer porous silicon (PSi) films were electrochemically anodized on monocrystalline, boron doped p + -type Si (100) wafers of 0.01-0.02 Ωcm resistivity. Electrolyte used in the process was a 1:1 (vol.) solution of hydrofluoric acid (HF, 38%) and absolute ethanol (EtOH). The anodization current profile repeated a cycle of low and high current density periods to create fracture planes on the PSi layer to assist in the milling process. The PSi multilayer was finally lifted off from the wafer as a freestanding film utilizing an electropolishing current pulse. The PSi films were initially stabilized by thermal carbonization using acetylene (C 2 H 2 ) as follows. The PSi films were dried under N 2 flow in a quartz tube for at least 30 min to remove residual moisture and oxygen at room temperature. Following the drying, acetylene was added to the gas flow at a ratio of 1:1 (vol.) N 2 /C 2 H 2 for 15 min at room temperature. Under S3 the N 2 /C 2 H 2 flow, the tube was placed in a furnace for 15 min at 500°C, after which the films were allowed to cool back to room temperature under N 2 flow. Finally, N 2 /C 2 H 2 flow was added at room temperature for 10 min before thermally annealing the films at 820°C for 10 min under N 2 flow. The obtained thermally carbonized PSi (TCPSi) films were then cooled back to room temperature under N 2 flow.
The non-stoichiometric SiC surface structure enabled functionalization of the TCPSi films through generation of -OH terminal groups on the surface by immersing the films briefly into HF solution (1:1 (vol.) HF/EtOH). 4 Following this, the films were silanized for 1 h using a 10 vol-% 3-(aminopropyl)triethoxysilane (APTES)  anhydrous toluene solution at room temperature. After removal of the excess silane, the films were dried for 16 h at 105°C. The aminopropylsilane-terminated TCPSi films (APSTCPSi) were then milled in a ball mill into nanoparticles using a 5 vol-% APTES-toluene solution as the milling medium. Size selection of the APSTCPSi nanoparticles was done with centrifugation using EtOH as the particle dispersant.

APSTCPSi Nanoparticles Characterization
The morphology of APSTCPSi was investigated by a transmission electron microscope (TEM, Tecnai F12, FEI Company, USA). The hydrodynamic diameter (Zaverage) and zeta-potential of the nanoparticles were analyzed by a Zetasizer Nano ZS (Malvern Instruments, UK) at 25 ℃. The porosity of the particle was analyzed with nitrogen sorption at 196 C using Tristar 3000 (Micromeritics Inc., USA). Specific surface area was calculated from the isotherm ( Figure S1) using Brunauer-Emmett-

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Teller method and the pore volume was estimated as the total adsorbed amount at a relative pressure of 0.97. Average pore diameter was calculated by assuming the pore shape as cylindrical.

APSTCPSi Nanoparticles Hydrolysis
To optimize the hydrolysis condition of APSTCPSi nanoparticles, 0.5 mg of nanoparticles were dried in the oven at 40 ℃ overnight. Then, the nanoparticles were incubated in the 0.1 M of NaOH solution at 60 ℃ water bath for different time-points (0.5, 1, 3, and 6 h). At each time-point, samples were collected and measured by a Zetasizer Nano ZS. Moreover, these samples were also investigated by ultravioletvisible absorbance at 250800 nm by a Varioskan Flash fluorometer (Thermo Fisher Scientific, USA). In addition, a digital camera was used to follow the general hydrolysis of the samples.

NMR Spectroscopy
Solution 1 H proton NMR data was collected by a Bruker Avance III 400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at 400 MHz.

APSTCPSi Nanoparticles PEGylation and Quantitative NMR Spectroscopy
APSTCPSi nanoparticles and mPEG-NHS were dissolved into 1 mL of HBSS (pH 7.4) with different molar ratios (1:0.1, 1:0.5, 1:1, and 1:2, amine:NHS) with constant stirring. Then the reaction was performed for 3 h at room temperature. After that, the nanoparticles were centrifuged for 5 min (15000g) and washed with deionized (DI) S6 water for three times. After drying overnight at 40 ℃ in the oven, these nanoparticles were dissolved in 800 μL of 0.1 M of NaOD/D 2 O with internal standard at 60 ℃ for 3 h and further analyzed, using the same qNMR protocol described above.

NMR Data Processing and Calculation of the NH 2 functional groups
All the NMR spectra were processed by MestReNova software. After Fourier transformation, the chemical shift was referenced by the residual water signal (a broad peak centered at 4.79 ppm). The phase was corrected automatically first, followed manual adjustment for specific peaks if the automatic correction was not satisfying. The baseline was corrected by fifth-order polynomial fit with manually adjusted filter.
To calculate the amine groups on APSTCPSi nanoparticles, we used the integral data from Figure 3a where, Area Internal standard and Area peak a are the integral area of the internal standard peak (4H from benzene ring of potassium phthalate) and peak a (2H from methylene protons next to the amine groups), N Internal standard is the molar amount of the internal standard, and N -NH2 is the molar amount of -NH 2 .

Elemental analysis of APSTCPSi Nanoparticles
Automatic elemental analyzer vario MICRO cube (HANAU Elementar Analysensysteme GmbH, Germany) was used for the determination of carbon as CO 2 , hydrogen as H 2 O, nitrogen as N 2 and sulfur as SO 2 in the sample. N 2 is not adsorbed in S7 the adsorption column and is the first measuring component to enter the thermal conductivity detector (TCD). The adsorption column is heated stepwise to desorption temperatures of the CO 2 (60 °C), H 2 O (140 °C) and SO 2 (210 °C). The samples were analyzed in triplicates and the results are shown in Table S1 below. (2)

NMR Data Processing and Calculation of the PEGylation functional groups
To calculate the PEG conjugated on the APSTCPSi nanoparticles, we used the integral data from Figure 4b, and calculated using Eq. where, Area Internal standard is the integral area of internal standard peak (4H from benzene ring of potassium phthalate), Area peak f is the integral area of PEG repeating unit (peak f, OCH 2 CH 2 O), 5000 is the molecular weight of the mPEG-NHS, 44 means the molecular weight of the repeating units, N Internal standard is the molar amount of the internal standard, and N PEG is the molar amount of PEG.

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However, because in the spectra shown in Figure 4b, peak f is overlapping with peak d (OCH 2 from the ethoxy group) and the integration was affected accordingly.
Then we made the integration of peak e (OCH 2 CH 3 from the same ethoxy group), and deduce the area of peak d from Area peak f+d by using Eq. (4): (4) Area peak f = Area peak f + d -