(229c) Convenient Generation and Detection of Oxygen Gradients to Investigate Hypoxia-Induced Effects on Cancer Cells

Recent studies have shown that hypoxia is a critical factor in the induction of drug resistance and angiogenesis in cancer cells. However, most current studies are performed either at the atmospheric levels (21% O₂) or utilize O₂ controlled incubators to maintain a defined O₂ stress, conditions that do not adequately reflect the microenvironment of solid tumor tissues. Recently, microfluidic approaches have also been proposed to effectively generate O₂ gradients but involve complex gaseous connections or indirect oxygen detection through secondary byproducts. Here, we demonstrate a novel approach for passive generation of linear oxygen gradients within microfluidic channels with simultaneous detection capability of real-time O₂ concentrations. This platform consists of just a single source of O₂ enriched and an O₂ depleted media to establish long lasting linear gradients ideal for investigating the hypoxic effects on cancer cells. The approach is based on the split and recombine strategy commonly utilized to create passive chemical gradients. The diffusion of dissolved oxygen through PDMS is prevented by creating a thin (5-10 µm) glass coating on the top three sides of the microchannels while excluding the bottom side to allow real-time detection through integrated Platinum (II) octaethylporphyrin ketone (PtOEPK) sensors. PtOEPK is dissolved in polystyrene-toluene (7% w/w) solution at 1 mg/ml concentration and spin-coated on a glass slide. The PDMS device is subsequently bonded to the oxygen-sensing layer after applying the three-sided sol-gel coating. Compared to the uncoated channels, the coated device exhibits superior dynamic oxygen sensing ability, with significantly larger changes in the detection intensity. Calibration studies reveal that the glass coated device follows a linear Stern-Volmer relationship, with a superior I₀:I₁₀₀ ratio as compared to bare PDMS. Fluorescent imaging confirmed successful generation of an oxygen gradient at flow rates of 10 nLmin^-1 and 0.1 µLmin^-1consistent with the COMSOL simulation results. This is the first study where gaseous concentration gradients have been established through passive diffusion without requiring active gaseous connections or cytotoxic chemicals. This unique approach offers versatile applications such as the creation of parallel oxygen and chemical gradients to investigate effects of hypoxia induced drug resistance on cancer cells.

Keywords: Hypoxia, oxygen sensing, microfluidics