The Development of a Whole Cell-Based Biosensor for Detecting Toxins: Response and Biocompatibility Studies

摘要

A novel whole-cell biosensor for simple, reliable, and quick screening of toxins has been developed. The sensor has multiple applications in medical, food, and environmental industries and in homeland security. 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 inhibiting the response of the ion-selective electrode (ISE). When the monolayer is exposed to agents that increase the permeability (e.g. toxins), ions can diffuse through the membrane, and a potential response from the ISE is achieved. Hence, the measured potential response provides an indirect measurement of the toxin. Histamine, a model toxin that increases the permeability of HUVEC monolayers, was used in this study. Endothelial cells were seeded to the membrane and allowed to spread and form a confluent monolayer over the membrane surface. The electrode response and sensitivity were measured for the following conditions: (1) the membrane without cells and without histamine, (2) the membrane without cells and with histamine (1x10-1M), (3) the membrane with cells and without histamine, and (4) the membrane with cells and with varying concentrations of histamine. The cell-based sensor was exposed to histamine for 20 min and then immediately tested for a potential response. The tests confirm the inhibited ion transport caused by the presence of a confluent HUVEC monolayer. The results also show that histamine alone does not affect the overall response of the ISE. When the cell-based membranes are exposed to varying concentrations histamine, the overall response increases with increasing histamine concentration. Thus, the measured potential is an indirect measurement of the histamine concentration. Further experiments were performed for a similar molecule, histidine, to test for selectivity. The cell permeability was unaffected by histidine, and the sensor response remained unchanged. In addition to toxin detection, this HUVEC-modified sensor platform may be useful in the design of biocompatible biosensors for detection in blood. Biocompatibility tests were performed using human platelet-rich-plasma (PRP) and a fluorescently labeled Annexin V protein that binds to activated platelets. Fluorescence micrographs of the surfaces revealed that platelets readily aggregated on the surface of both of the control membranes containing no RGD or HUVECs. Following RGD immobilization, the hemocompatibility was greatly improved, but platelets still aggregated on the membrane surface. Finally, the CTA membranes with attached RGD and HUVECs illustrated more improved biocompatibility versus the controls. Further experiments will be conducted in order to confirm the preliminary results.