20.109(S11):WF Red Research Proposal

Detection of Pathogenic Bacteria Using Bacteriophage Virus Biosensors
Paula C. Trepman, Sabina Sood

Statement of Purpose
Utilizing T4-bacteriophage viruses, we want to develop a biosensor that can detect pathogenic bacteria, such as Escherichia coli. Previous research has found that Dithiobis(succinimidyl propionate) (DTSP) self-assembled monolayers can covalently attach T4 phages, immobilizing them on the surface. This property can be exploited by creating a gold/DTSP/T4-phage chip which selects for a particular type of bacteria that is dependent on the T4 bacteriophage. By incorporating the chips into a bisensor, the sensor can be employed for food and water safety assessment and testing for bacterial contamination of blood transfusions.

Background
Surface plasmons that are extremely sensitive to surface changes, including the adsorption of molecules on the sensor surface, can be used in biosensor technology. By reflecting light of a specific wavelength off the side of the chip, a certain reflected wave is produced. In this study, utilization of a cysteine and cysteamine self-assembling monolayer, Dithiobis(succinimidyl propionate) (DTSP), effectively attached bacteriophages to the gold chip, allowing for successful capture of specific bacteria. When bacteria bound to bacteriophage virus, this altered the refractive index of the chip, thereby indicating the presence of the bacterium. A gold/DTSP/T4 bacteriophage chip was utilized for specific capture of k12 E. coli Additionally, it was determined that the chip can be reused by treatment in NaOH. (Sunil et al--see resources)

A phage-based magnetoelastic biosensor was utilized to detect Salmonella on the surfaces of fresh tomatoes. The biosensor was comprised of a magnetoelastic biosensor platform covered in E2 phage. The resonance oscillation and frequency were then determined through magnetic field detection. A statistically significant shift in resonance frequency was observed in the experimental biosensors versus the control and binding specifity of the biosensor was verified by scanning electron microscopy, indicating the effectiveness of the magnetoelastic biosensor in sensing Salmonella. (Li-- see resources)

Bacterial contamination of blood occurs at a rate of 1 in 2,000 to 3,000 platelet units and 1 in 30,000 Red Blood Cell units. In a study of 38 clinically proven severe reaction to transfusion with contaminated blood, isolated bacteria included Enterobacteriaceae, Pseudomonas aeruginosa, Staphylococcus aureus, coagulase-negative staphylococci, streptococci, and Bacillus species. The authors stated that current methods of bacterial detection are too insensitive on new platelets or have high false-positive detection rates. As 42 percent of these severe blood contamination cases resulted in patient death, clearly an effective, fast, low-cost, and accurate method to test blood for bacteria would be useful within the medical field. (Jacobs et al--see references).

Protocol

 * 1) Produce a gold chip
 * 2) Create a DTSP monolayer on the chip
 * 3) Cover the chip with a bacterium-specific bacterophage solution
 * 4) Test for binding of bacteria to the chip by surface plasmon resonance

Resources
Chemically immobilized T4-bacteriophage for specific Escherichia coli detection using surface plasmon resonance

Direct detection of Salmonella typhimurium on fresh produce using phage-based magnetoelastic biosensors

Don’t bug me: the problem of bacterial contamination of blood components—challenges and solutions

Analysis of Bacterial Detection in Whole Blood–Derived Platelets by Quantitative Glucose Testing at a University Medical Center