Size, Surface Charge, and Morphology of NPs

The hydrodynamic particle size of the SiNP was determined using nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). For NTA measurements, the different SiNPs were diluted to a concentration of 10 µg/mL with deionized water and analyzed using the NanoSight LM10 instrument (Malvern, Kassel, Germany) and the NTA 3.2 Dev Build 3.2.16 software (Malvern Panalytical, Malvern, UK). For the measurements, the standard measurement protocol was followed with 5 captures per measurement and 15 s capture duration. The hydrodynamic sizes and zeta potentials of the SiNPs were further confirmed by DLS. A concentration of 100 µg/mL of NPs was prepared in the buffer used for conjugation and the measurements were performed using the ZetaSizer Nano ZS (Malvern Panalytical, Malvern, UK) and the ZetaSizer Software (Malvern, 7.03, Malvern Panalytical, Malvern, UK), modulating the settings for refractive index of the NP composition and dispersant (1.45 for silica).

The primary particle size of the SiNPs was determined by transmission electron microscopy (TEM). For measurement, 2 µL of a 10 µg/mL NP dispersion was dried overnight on a lacy carbon-coated copper TEM grid and imaged using the JEM F200 (JEOL, Freising, Germany) electron microscope in TEM mode operated at 200 kV. Primary particle size was determined by calculating the mean ± SD of minimum 10 particles via image processing with the ImageJ software (NIH, Bethesda, MD, USA) and manual measuring.
