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 5×103 and 5×102 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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Greenberg, D.L., Busch, J.D., Keim, P.D. & Wagner, M. Identifying experimental surrogates for Bacillus anthracis spores: a review. Investigative Genetics 1, 1–12 (2010).
Hutchison, J.R. et al. Reagent-free and portable detection of Bacillus anthracis spores using a microfluidic incubator and smartphone microscope. Analyst 140, 6269–6276 (2015).
Weller, S.A. et al. Evaluation of two multiplex realtime PCR screening capabilities for the detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples generated from murine infection models. J. Med. Microbiol. 61, 1546–1555 (2012).
Janzen, T.W. et al. Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense. J. Food. Prot. 78, 355–361 (2015).
Létant, S.E. et al. Rapid-viability PCR Mmethod for detection of live, virulent Bacillus anthracis in environmental samples. Appl. Environ. Microbiol. 77, 6570–6578 (2011).
Rasko, D.A. et al. Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation. Proc. Natl. Acad. Sci. USA 108, 5027–5032 (2011).
Thierry, S. et al. A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis. J. Microbiol. Methods 95, 357–365 (2013).
Summerer, D. et al. A flexible and fully integrated system for amplification, detection and genotyping of genomic DNA targets based on microfluidic oligonucleotide arrays. New. Biotechnol. 27, 149–155 (2010).
Zwick, M.E. et al. Genomic characterization of the Bacillus cereus sensu lato species: Backdrop to the evolution of Bacillus anthracis. Genome Res. 22, 1512–1524 (2012).
Morel, N. et al. Fast and sensitive dn of Bacillus anthracis spores by immunoassay. Appl. Environ. Microbiol. 78, 6491–6498 (2012).
Wang, D.B. et al. Detection of B. anthracis spores and vegetative cells with the same monoclonal antibodies. PLoS One 4, e7810 (2009).
Li, B., Yua, Q. & Duan, Y. Fluorescent labels in biosensors for pathogen detection. Crit. Rev. Biotechnol. 35, 82–93 (2015).
Ryu, J., Lee, E., Lee, K. & Jang, J. A graphene quantum dots based fluorescent sensor for anthrax biomarker detection and its size dependence. J. Mater. Chem. B 3, 4865–4870 (2015).
McGovern, J.P. et al. Label-free flow-enhanced specific detection of Bacillus anthracis using a piezoelectric microcantilever sensor. Analyst 133, 649–654 (2008).
Cho, J.H., Han, S.M., Paek, E.H., Cho, I.H. & Paek, S.H. Plastic ELISA-on-a-chip based on sequential cross-flow chromatography. Anal. Chem. 78, 793–800 (2006).
Han, S.M. et al. Plastic enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor for botulinum neurotoxin A. Anal. Chim. Acta 587, 1–8 (2007).
Seo, S.M. et al. Food contamination monitoring via internet of things, exemplified by using pocket-sized immunosensor as terminal unit. Sensor. Actuat. B 233, 148–156 (2016).
Cho, I.H., Paek, E.H., Kim, Y.K., Kim, J.H. & Paek, S.H. Chemiluminometric enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor based on cross-flow chromatography. Anal. Chim. Acta 632, 247–255 (2009).
Cho, J.H., Paek, E.H., Cho, I.H. & Paek, S.H. An enzyme immunoanalytical system based on sequential cross-flow chromatography. Anal. Chem. 77, 4091–4097 (2005).
Zasada, A.A., Forminska, K., Zacharczuk, K., Jacob, D. & Grunow, R. Comparison of eleven commercially available rapid tests for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis. Lett. Appl. Microbiol. 60, 409–413 (2015).
Wang, D.B. et al. Detectionof Bacillus anthracis sporesbysuper-paramagnetic lateral-flowimmunoassaysbasedon “RoadClosure”. Biosens. Bioelectr. 67, 608–614 (2015).
Waller, D.F., Hew, B.E., Holdaway, C., Jen, M. & Peckham, G.D. Rapid detection of Bacillus anthracis spores using immunomagnetic separation and amperometry. Biosensors 6, 61; doi:10.3390/bios6040061 (2016).
About this article
Cite this article
Han, SM., Kim, YW., Kim, YK. et al. Performance Characterization of Two-Dimensional Paper Chromatography-based Biosensors for Biodefense, Exemplified by Detection of Bacillus anthracis Spores. BioChip J 12, 59–68 (2018). https://doi.org/10.1007/s13206-017-2108-9
- Bioterrorism agent
- Field-version of ELISA
- Two-dimensional chromatographic assay
- Chemiluminometric detection
- Cooled charge-coupled device