| A novel whole-cell potentiometric biosensor for screening of toxins has been developed. The constructed biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) attached to an ion-selective cellulose triacetate (CTA) membrane modified with a covalently attached RGD (arginine-glycine-aspartic acid) peptide sequence. When the HUVECs form a confluent monolayer, ion transport is almost completely inhibited, thereby reducing the response of the ion-selective electrode (ISE). When the monolayer is exposed to agents that increase its permeability (e.g., toxins), ions can diffuse through the membrane, and a potential response from the ISE is achieved. Histamine, a model toxin that increases the permeability of HUVEC monolayers, was first used in this study. When the cell-based membranes were exposed to varying concentrations of histamine, the overall response was obtained as an indirect function of the histamine concentration. Further experiments were performed to optimize the exposure time of the biosensor to histamine and to determine the mechanism of the biosensor response. Similarly, the biosensor responded to the presence of vascular endothelial growth factor (VEGF). VEGF is a cytokine and endothelial cell (EC) mitogen that has been studied for its role in angiogenesis of malignant tumors. VEGF is elevated in the serum and plasma of cancer patients. When the cell-based membranes were exposed to physiological concentrations of VEGF, the ISE response increased with increasing VEGF concentration and hence, a relationship between the potential value and VEGF concentration could be achieved. The biosensor was then exposed to media samples obtained from cultured human melanoma cells. A VEGF ELISA confirmed that the biosensor model closely predicted the VEGF concentrations produced by the cancer cells. Furthermore, hemocompatibility tests were performed on the cell-based biosensor to determine its usability with blood samples. Platelet activation and aggregation was determined as a function of the cell spacing via fluorescence microscopy. Finally, as part of a NASA internship, ion-selective sensors were miniaturized for use in environmental sensing arrays. This study has direct application to the cell-based biosensor as future studies will focus on the miniaturization of the cell-based biosensor into a planar form for simultaneous detection of multiple toxins. |
