2.2. Chemical Modification of Silica Particles

The silica spheres are highly porous as delivered, with a pore volume of 0.9 mL/g, consisting of greater than 97% open pores and a mean pore size of 11 nm. This porosity was utilized for fluorescently dyeing not just the surface but also the interior of the particles. A modified Stöber synthesis was used to covalently bind the dye to the particles.³⁸⁻⁴⁰ A detailed description of the synthesis is given in the Supporting Information, section 1.1. Wenzl et al. recently used a very similar procedure to prepare the same Kromasil silica microspheres for their confocal study.⁴¹ The modified Stöber synthesis did not lead to a change in particle shape, as all of the particles are still perfectly spherical, as shown in Supporting Information Figure S1.a. The dyeing process does not lead to a significant change in particle size, with the mean particle diameter being d_50,3 = 6.43 ± 0.03 μm after treatment (Supporting Information Figure S2). BET (Brunauer–Emmett–Teller adsorption method) surface measurements show a reduction from 295 to 249 m²/g. Therefore, although the porosity of the dyed particles has decreased, it remains very high.

Additional reaction steps were completed using the dyed porous silica particles to further modify the particle surface. An overview of the prepared particle types used in this study is shown in the Supporting Information in Table S1, including both porous and nonporous particles. To produce nonporous particles, the modified Stöber reaction was repeated using small numbers of dyed particles, with some differences in the reaction scheme: no more coupled dye was added, and higher concentrations of tetraethyl orthosilicate (TEOS) were used (3 mL TEOS per gram of dyed silica spheres). The measured particle density remained nearly unchanged during all of the reactions at 2.04 ± 0.07 g/mL. Particle densities have been measured at 20 °C using helium gas pycnometry (MultiVolume Pycnometer 1305, Micromeritics Instrument Corporation, Norcross, GA, USA), and the results were cross-checked by liquid pycnometry in water.

To change the contact angle, the silica surface of either porous or nonporous particles was treated with trimethyl chlorosilane (TMCS, Alfa Aesar, Karlsruhe, Germany). When exposed to TMCS, the −OH groups on the silica surface are replaced by trimethylsilyl groups (−Si–(CH₃)₃), making the particle surface more hydrophobic.⁴² A detailed description of the completed method is given in section 1.3 of the Supporting Information.