Rapid Detection and Discrimination of Bacillus Species Using IMS-SERS

Abstract

Bacillus species are gram positive, spore-forming bacteria which include B. anthracis, a class A bioterrorism agent according to the Centers for Disease Control and Prevention. Because of anthrax’s high toxicity, it is important to be able to quickly and accurately detect B. anthracis spores. Immunomagnetic separation (IMS) has previously been shown to capture toxins from complex media, such as food. Subsequent detection with Surface-Enhanced Raman Spectroscopy (SERS) has been shown to be highly sensitive for detection and discrimination of other toxins. This research aimed to discriminate three different Bacillus species and establish a procedure based on IMS-SERS that can detect B. anthracis in under 20 minutes.

We found that the Bacillus species could be differentiated when SERS spectra were analyzed using principal component analysis; discrimination between cell states could be achieved with SERS when using hierarchical cluster analysis. The limit of detection was 2*104 spores/mL due to inconsistency of the raman signal as a result of incomplete coverage of the silver dendrites. In order to achieve a lower limit of detection, the spores were treated with dodecylamine, which rapidly digests the sporal coat, following IMS and adds little time to sample preparation. Upon digestion, the spore biomarker dipicolinic acid (DPA) is released. DPA is a highly raman-active compound, which allows for use as a raman marker and due to its high abundance in the spores, allowed for a lower limit of detection at 1* 104 spores/mL. Based on published toxicological data, detection at these limits is sufficient for protecting the public in case contamination of food.