Simultaneous Detection of Salmonella typhimurium and Bacillus anthracis Spores Using Phage-Based Magnetoelastic Biosensors S. Huang 1 , H. Yang 1 , M. Johnson 1 , I. Chen 2 , V. A. Petrenko 3 , and B. A. Chin 1 1. Materials Engineering, Auburn University, Auburn, AL 36849, USA 2. Dept. of Botany & Microbiology, Auburn University, Auburn, AL 36849, USA 3. Dept. of Pathobiology, Auburn University, Auburn, AL 36849, USA ABSTRACT The objective of this research was to investigate simultaneous detection of two different biological pathogens using one type of sensor. The biosensors investigated are comprised of a magnetoelastic (ME) particle coated with phage as the biomolecular recognition element, and resonated by an AC magnetic field. As cells/spores are captured by the phage, the mass of the sensor increases, which results in a decrease in the sensor’s resonant frequency. Two genetically engineered phages, binding with S. typhimurium or B. anthracis, were immobilized onto two separate ME particles with different dimensions, allowing simultaneous measurement of two different resonance peaks. Upon exposure to solutions containing known concentrations of each analyte, only the biosensor coated with the corresponding phage responded. SEM and TEM were used to verify and quantify interaction of each biosensor with its target analyte. Results show that the observed number of bound cells/spores corresponds closely to the number calculated from the frequency shift data. Keywords: magnetoelastic, biosensor, multiple, phage, Salmonella typhimurium, and Bacillus anthracis spores 1. Introduction Every year, over 76 million Americans suffer from food-borne illnesses, out of which about 10,000 result in death. Bacterial pathogens account for more than 50% of these food-borne illnesses, of which S. typhimurium is one of the most common. Additionally, spores and fungi affect the quality of our food leading to spoilage of the product. Recently, fears of deliberate contamination of our food supply have become a concern, B. anthracis (anthrax) being a primary pathogenic spore of interest. Intensive research has been focused on developing techniques for early detection of these pathogens. The need for new technologies that can be used in the field is great since traditional laboratory based methods are time-consuming and manpower intensive, usually requiring several days to yield results [1]. Additionally, for most of the current microbiological tests, the testing procedure is complicated, equipment is expensive and qualified operators are required. Therefore, the development of portable, rapid, specific, and sensitive biosensors for real-time, field detection is now a primary need. In this research, we investigate the use of ribbons of magnetoelastic (ME) material as the sensor platform. A bio-molecular recognition probe (phage that binds S. typhimurium bacteria or B. anthracis spores) is immobilized onto the platform surface. It should be noted that in the experiments we use Bacillus anthracis Sterne spores, the nonpathogenic vaccine strain, which is almost identical to the pathogenic strain of Bacillus anthracis and has similar binding characteristics. The objective of these experiments is to investigate whether it is possible to simultaneously detect two different types of biological pathogens using this method. 2. Materials and Methods 2.1 Magnetoelastic Platform Commercially available ME strips (2826MB Metglas TM film, Fe 40 Ni 38 Mo 4 B 18 , Honeywell) were polished to a thickness of 15μm, followed by cutting using a micro- dicing saw. Sensors were diced into two sizes of 2.0 x 0.4 mm and 1.9 x 0.4 mm in order to create two sensors with two distinct, different resonance peaks. These platforms were rinsed with fresh acetone, methanol, and finally dried in air to remove the adhesive used in the dicing process. Prior to further processing, the clean platforms were annealed to remove residual stresses, as well as any remaining trace organic residue. Thin films of chromium and then gold were deposited onto the surfaces of the ME platforms using a Denton™ Vacuum Discovery-18 sputtering system (Moorestown, NJ). The gold layer is necessary for bioactivity, as well as corrosion resistance, while chromium serves as an interlayer between the gold and the Metglas™ material for improved adhesion. 2.2 Bacteriophage Immobilization To form the biosensor, the two different sizes of sputtered ME platforms were immersed into two genetically engineered phage solutions (produced by Dr. Petrenko [2,3] of Auburn University’s college of Veterinary Medicine) for 1 hour, allowing the phage to attach to the sensors’ surface. One phage is for binding with S. typhimurium and the other is for binding with B. anthracis. Afterwards, the loose binding of phage, as well as the salts contained in phage solution, were subsequently rinsed in distilled water, and NSTI-Nanotech 2007, www.nsti.org, ISBN 1420061836 Vol. 2, 2007 515