Performance Characterization of Two-Dimensional Paper Chromatography-based Biosensors for Biodefense, Exemplified by Detection of Bacillus anthracis Spores

Bacillus anthracis ( B. anthracis ), the causative agent of anthrax disease, is a Gram-positive spore-forming bacterium which can be used as a threatening bioterrorism agent. We developed enzyme-linked immunosorbent assay (ELISA)-on-a-chip biosensors for rapid, sensitive analysis of B. anthracis spores based on two-dimensional, cross-flow chromatography. In order to establish optimal assay conditions, a polyclonal antibody and four monoclonal antibodies against B. anthracis were raised and examined to characterize their analytical sensitivity as well as specificity. The biosensor results showed that a monoclonal antibody pair not only offered a relatively low detection limit for B. anthracis compared to other antibody combinations, but also displayed no cross-reactivity with other microorganisms belonging to the Bacillus genus. For detection of ELISA enzyme signal (e.g., horseradish peroxidase), chemiluminescent detection in combination with cooled charge-coupled device enhanced the sensor performance in terms of assay time, compared to that achieved by colorimetry. Under optimal conditions, the biosensor was able to detect a minimum threshold of 5×10 3 and 5×10 2 spores/mL for two different B. anthracis strains, NCCP 12860 (Sterne) and NCCP 10666 (Haman #1), respectively. Furthermore, the chemiluminometric sensor was minimally affected by the presence of potential interferents in samples such as baby powder, skim milk, and sucrose, indicating its potential utility for the analysis of bioterrorism agents directly in the field.