Capture and Detection of Bacillus anthracis Spores Using Aptamer Based Surface Enhanced Raman Spectroscopy

Introduction: Bacillus anthracis is a highly pathogenic spore forming bacterium and a class A potential bioterrorism agent. Since spores of B. anthracis can survive pasteurization, it is considered a major threat for food defense, as well as in US mail threats, and its detection in is critical for preventing a potential terrorist attack.  

Purpose: The goal of this project was to evaluate a method for the capture and detection of spores of B. anthracis in foods/dry powders quickly with high accuracy, (#spores/ml). In this study, we demonstrate a system which combines aptamer based capture and Surface Enhanced Raman Spectroscopy (SERS) using a silver dendrite nano-surface as a method of signal enhancement. This methodology has been previously shown to detect ricin in foods such as milk and orange juice.

Methods: Two published aptamer sequences (BAS-6F and BAS-6R) were constructed. These are single strands of DNA sequences, forming unique structures that bind to surface proteins of B. anthracis. The aptamers were initially bound to silver dendrites and then used to capture the spores from solution. The coverage of the aptamers on silver dendrites was optimized. SERS Raman spectra before and after capturing spores were acquired and by principle component statistical analysis (PCA).

Results: Plate count determination (#spores/ml) and spectral analysis indicated that both aptamers effectively captured B. anthracis spores from water. Plate counts found a capture efficiency of 70-80%. Using the superior BAS-6F aptamer, the limit of detection was 10³-10⁴ spores in water and 10⁴ spores in orange juice. We could discriminate between B. anthracis and B. mycoides spores.

Significance: This aptamer-capture-SERS process can be completed in less than 40 minutes, which makes it a rapid method that can be used in food security and potential mail bioterror threats.