Label-free impedimetric electrochemical aptasensor for the detection of Bacillus anthracis spores using a disposable screen-printed electrode.

Anthrax is an acute zoonotic disease caused by a bacterium, Bacillus anthracis. It continues to pose a global public health and biodefense concern, with recurring outbreaks and long-lasting soil contamination reported worldwide. Anthrax remains a significant zoonotic threat in several regions of India, particularly Karnataka, Andhra Pradesh, Maharashtra, and Tamil Nadu. Nearly 90 confirmed outbreaks were reported between 2018 and 2023 across 14 districts in Karnataka, predominantly affecting sheep and cattle. The persistence of B. anthracis spores in endemic soils demonstrates an urgent need for a rapid, field-deployable, and reliable diagnostic platform. In this study, an aptamer-based impedimetric electrochemical biosensor was evaluated using a disposable screen-printed gold nanoparticle electrode/chip (DEP) as a sensing platform for the detection of B. anthracis spores. The electrode was functionalized with 11-mercaptoundecanoic acid (MUA), EDC-NHS and the aptamer layer was characterised using electrochemical impedance spectroscopy (EIS). The charge-transfer resistance (Rct) increased progressively with each modification step, validating successful surface chemistry and aptamer immobilization. Endospores were prepared using Schaeffer Sporulation Agar, achieving >95% purity as confirmed by Gram and Schaeffer-Fulton staining. In parallel, sporulation of Bacillus thuringiensis produced oval spores and bipyramidal parasporal crystals, confirmed by Amido Black staining, enabling clear morphological differentiation from B. anthracis. The biosensor exhibited high specificity toward B. anthracis spores, with minimal response to B. thuringiensis as negative controls. EIS demonstrated a clear concentration-dependent increase in Rct across 20 - 500 spores/mL. A strong correlation was observed between Rct and spore concentration (R2 = 0.897, p < 0.05), with a well-defined linear response between 20 and 200 spores/mL. At higher concentrations, a sharp rise in impedance was likely due to spore aggregation and increased surface blocking on the electrode. Overall, the platform provides a rapid, label-free, and sensitive method for detecting B. anthracis spores, highlighting its potential utility in biodefense, environmental monitoring, and field-level diagnostics.