The microparticles were prepared in a simple two-step process. The first step consists of electrostatic immobilization of the oxygen-sensitive fluorophore, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride (Ru), and the reference fluorophore, Nile blue chloride, onto ~10 Ám silica gel particles. Ru was chosen because of its high quantum yield, high oxygen quenching, and thermal stability at physiological temperature. The reference fluorophore allows ratiometric (or normalized) measurements that correct for the typical drawbacks of fluorescence intensity measurements such as bleaching, heterogeneous fluorophore concentration, fluctuations in excitation light intensity, and camera sensitivity. The second step consists of the encapsulation of the fluorophore-bound silica gel particles within larger poly(dimethylsiloxane) (PDMS) particles. PDMS is optically transparent, hydrophobic, biocompatible, and highly permeable to gases such as oxygen, nitrogen, and carbon dioxide.
To characterize the microparticles, we measured particle size distribution and compared the performance of the sensing microparticles to that of a conventional dissolved oxygen meter. The 10-40 Ám size of the microparticles makes them versatile, allowing them to be suspended in a large range of biomaterial substrates. The microparticle response time was approximately 37% faster than that of a conventional dissolved oxygen meter. Microparticle sensor response was fully reversible and no evidence of hysteresis was observed. Finally, microparticle calibration shows that it is possible to measure oxygen partial pressures in ranges relevant to tissues culture studies and in close proximity to the cells. Sample size is only limited by scaffold transparency and the microscope depth of field; herein, transparent hydrogel samples 1.5 cm wide x 3.8 cm long x 260 Ám deep were sampled. A measurement of one ~2 x 2 mm field of view can be performed in about 10 s. The versatility of the described sensing microparticles will allow for future studies where dynamic spatial correlations between cell function and oxygen concentrations can be achieved reliably both in two-dimensional and three-dimensional in vitro environments.