(376d) Biosensor Incorporating Cell Barrier Architectures for Detecting Staphylococcus Aureus Alpha Toxin

Staphylococcus aureus is one of the most common bacterial pathogens involved in various infections like septicemia, endocarditis, osteomyelitis, septic arthritis etc. The fact that S. aureus is the major cause of postoperative infections and also infections associated with prosthetic devices makes it one of the most important pathogens in nosocomial setting. Isolation and cultivation of the pathogen and determination of antibodies against different antigens of S.aureus are the major diagnostic methods for different staphylococcal diseases. However, the major disadvantages associated with these methods are the lack of sensitivity and the time involved for diagnosis. Hence, there is a need for other methods that would provide quick and reliable diagnosis at the early stages of staphylococcal diseases. Alpha toxin, a protein exotoxin, produced by a majority (85-90%) of S. aureus strains, plays a critical factor in S.aureus induced pathogenicity. As a matter of fact, the presence of alpha toxin has been implicated to be a necessary criterion for the biofilm formation by S.aureus. This virulent factor of S.aureus has been shown to exhibit haemolytic, cytotoxic, dermonecrotic as well as lethal effects on laboratory animals. The cytotoxicity of alpha toxin is attributed to its transmembrane pore forming ability leading to the leakage of ions and macromolecules which eventually results in necrosis. Development of an enzyme immunoassay (EIA) with monoclonal antibodies as well as double antibody sandwich ELISA for detection of staphylococcal alpha toxin had been documented. Depending on the methods employed, the assays require 6 hours to overnight incubation for effective determination of alpha toxin. The present study is focused towards detection of staphylococcal alpha toxin using a whole cell based biosensor. The biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) seeded on to the surface of asymmetric cellulose triacetate (CTA) membrane of an ion selective electrode. This sensor takes advantage of endothelial cell permeability dysfunction to detect the presence of small quantities of permeability modifying agents. Earlier studies with this electrode have shown that a complete inhibition of the ion transport across the membrane is observed when a confluent monolayer of HUVECs is formed. This results in a reduced ISE response. When the biosensor is exposed to environmental and physiological toxins that effect cell permeability, the response of the biosensor serves as an indirect measurement of the presence of toxin. Previous published studies have shown that the sensor can be used to measure the presence of a model toxin, histamine and we have recently extended this work to the detection of vascular endothelial growth factor (VEGF). Specifically, HUVECs were seeded onto the CTA membrane of an ISE with a seeding density of 1 x 105 cells/ml. The cells were allowed to spread and form a confluent monolayer over the membrane surface for 24 h at 37oC in a humidified incubator with 5% CO2. Following confirmation of a confluent monolayer, the electrode response to a known concentration of K+ ions was measured following treatment with 1000 ng/ml of staphylococcal alpha toxin over a time ranging from 10-90 mins. The results indicated that the sensor responded to the alpha toxin after 20 mins of exposure time. In addition to determine the detection limit of alpha toxin, the sensor was exposed to 0.1 ? 1000 ng/ml alpha toxin solution. The detection limit of this sensor for alpha toxin was found to be 0.1 ng/ml. Considering the fact that the acute toxic concentration of alpha toxin in human is 100-250 ng/ml of whole blood, it is hypothesized that this sensor, with its ability to detect 0.1ng/ml alpha toxin in 20 mins, has the ability to act as a diagnostic tool during the onset of staphylococcal diseases. The response of the sensor to the presence of alpha toxin can be attributed to the formation of intercellular gaps. The effect of alpha toxin on cell shape, gap formation and gap size distribution will be discussed.