Development and Study of Phage-Coated Magnetoelastic Biosensors for the Detection of Bacillus Anthracis Sterne Spores

Abstract

Bacillus anthracis spores have long been recognized as biological warfare agents because of their ability to cause mortality in humans and their long life under unfavorable environments. The early detection of Bacillus anthracis spores in very small amounts or very low concentrations is critical for national security. In this research a biosensor for the detection of Bacillus anthracis spores was developed that combines phage display technique with a magnetoelastic, wireless, detection platform. The affinity based biosensor utilizes a phage-derived diagnostic probe as the bio-molecular recognition element to capture target agents multivalently. Upon binding of the target agent to the sensor surface, the resonance frequency of the magnetoelastic biosensors decreases due to the additional mass of the target agent. Scanning electron microscopy was used to confirm the binding of spores to the sensor surface. The sensitivity of magnetoelastic acoustic sensors of size 5mm × 1mm × 20 µm was tested to be 130Hz per order of magnitude of spore concentration with a detection limit of 103 cfu/ml. The specificity and longevity of the sensors were also studied. To increase the sensitivity and detection limit of magnetoelastic biosensors, free standing, magnetoelastic resonating particles, as small as 500×100×4 µm and 200 × 40 × 4 µm, have been made using microelectronics fabrication techniques. The biosensors were tested in Bacillus anthracis spore solutions with concentrations from 102 to 108 cfu/ml. The detection limits and sensitivities of the sensors were determined based upon resonance frequency measurements. The study on the binding kinetics have shown that the dissociation constant (Kd) and the binding valency in Bacillus anthracis spore solutions are 193 cfu/ml and 2.32 for sensors of 500×100×4 µm, and 102 cfu/ml and 1.95 for sensors of 200×40×4 µm. To explore the possibility of the ultimate goal of single spore detection, a microfluidic chip was designed, fabricated and tested together with 200 × 40 × 4 µm sized magnetoelastic particles. This phage-coated magnetoelastic particle was introduced into the test chamber of the microfluidic chip and spore capturing experiments were conducted in the chamber. The change in the resonant frequency and the surface SEM micrographs of the particle confirmed the binding and detection of small numbers of spores. In summary, a rapid, specific and sensitive biosensor based on the techniques of magnetoelastic material and filamentous phage was investigated and demonstrated to be suitable for continuous environmental monitoring. With further developments in the fabrication techniques for nanoparticles, this sensor platform may be able to detect single spore.