Rapid measurements of 2,8-dihydroxyadenine (2,8-DHA) with a nanostructured electrochemical sensor in 5-fold diluted supernatants of endothelial cells exposed to oxidative stress

Abstract

Fast scan cyclic voltammetry (FSV) with a nanostructured carbon fiber sensor (N-CFS) was developed for direct measurements of the purine metabolite 2,8-dihydroxyadenine (2,8-DHA; i.e. 6-amino-1H-purine-2,8-dione) in endothelial cell supernatants as a marker of cell stress. The 2,8-DHA was measured in the supernatant of aortic (AECs) and pulmonary artery endothelial cells (PAECs), which were maintained in Hank's Balanced Salt solution (HBSS) and exposed to physiological oxygen pressures as well as to oxidative stress, hypoxia (specifically 3% O₂ for AECs) and hyperoxia (20% O₂ for PAECs). Dilution of the supernatants with phosphate buffer in the ratio of 1 : 5 allowed the optimization of FSV measurements with the N-CFS in cell supernatants.

The LOD for 2,8-DHA was 1 μM and the LDR was 2–15 μM with the sensitivity of (0.34 ± 0.01) nA μM⁻¹ (R² = 0.99). The changes in 2,8-DHA concentration when the cells were exposed to stress confirm that PAECs can adapt to stress. However, the results also show that the tolerance of AECs to hypoxia is low. Cellular pathways involved in the response of PAECs and AECs to oxidative stress are outlined.