Electrochemical Immunodetection of Bacillus anthracis Spores

Abstract

The Centers for Disease Control and Prevention (CDC) classifies Bacillus anthracis as one of the most dangerous pathogens that may affect public health and national security. Due to its importance as a potential biological weapon, this bacteria has been classified in the highest category A, together with such pathogens as variola virus or botulinum neurotoxin. Characteristic features of this pathogen that increase its military importance are the ease of its cultivation, transport, and storage and its ability to create survival forms that are extremely resistant to environmental conditions. However, beyond bioterrorism, B. anthracis is also a naturally occurring pathogen. Anthrax outbreaks occur in livestock and wildlife, particularly in spore-contaminated regions of Africa, Asia, and North America. Spores persist for decades, leading to recurrent infections and zoonotic transmission through direct contact, inhalation, or consumption of contaminated meat. This work presents a new electrochemical method for detecting and quantifying B. anthracis in spore form using a selective immune reaction. The developed method is based on the thiol-modified electrodes that constitute the sensing element of the electrochemical system. Tests with the B. anthracis spore suspension showed that the detection limit for this pathogen is as low as 10³ CFU/mL. Furthermore, it was possible to quantify the analyte with a sensitivity of 11 mV/log (CFU/mL). Due to several features, such as low unit cost, portability, and minimal apparatus demands, this method can be easily implemented in field analyzers for this pathogen and provides an alternative to currently used techniques and devices.

Keywords: Bacillus anthracis spore detection; electrochemical biosensor; immunoelectrochemical detection; microbiological analysis; portable sensor.

Figure 1

Modification of gold electrode: schematic illustration of the stages of modification. Cleaning of the gold electrode surface, formation of a self-assembled monolayer using 4-tert-butylbenzenethiol, deposition of gold nanoparticles, covalent immobilization of primary monoclonal antibodies specific to Bacillus anthracis, and passivation with bovine serum albumin to prevent nonspecific adsorption.

Figure 2

The Principal Component Analysis (PCA) biplot describing the specificity of the tested monoclonal antibodies against Bacillus spores and the differences between antibodies.

Figure 3

Cyclic voltammograms recorded for individual stages of modification of the working electrode surface: (a) electrochemical cleaning of the electrode; (b) working electrode with a layer of TBBT; (c) working electrode with a reconstituted layer of gold nanoparticles; (d) working electrode with deposited primary antibodies SA27.

Figure 4

Cyclic voltammograms after incubation with B. anthracis. The measurement was performed using a concentration of 10⁸ CFU/mL of B. anthracis spores in PBS. Five CV cycles were conducted for each electrode.

Figure 5

A graph showing the change in the recorded voltammogram depending on the concentration of B. anthracis from 10³ to 10⁸ CFU/mL in 0.1 M PBS at pH 7.4 and 1 mM of ferrocyanide.

Figure 6

A plot showing the relationship between voltage and current changes as a function of B. anthracis concentration from 10³ to 10⁸ CFU/mL in 0.1 M PBS and 1 mM of ferrocyanide at pH 7.4: current vs. concentration (a), potential vs. concentration (b). Each point corresponds to the current and potential values at the maximum of the reduction peak.

Figure 7

The scan of the electrode surface obtained using the CSLM (a) without antibodies, (b) coated with primary and secondary antibodies. The fluorescent image from the CSLM (c) without antibodies, (d) coated with primary and secondary antibodies. Image obtained using the STEM, magnification (1000×), (e) without antibodies, (f) coated with primary and secondary antibodies.