Colorimetric aptasensor for detection of Bacillus cytotoxicus spores in milk and ready-to-use food

Abstract

The high incidence of foodborne diseases caused by pathogenic bacteria raises concerns worldwide and imposes considerable public healthcare challenges. This is especially observed with dormant spores of Bacilli, which can often survive treatments used by the food industry to kill growing bacteria. The early and rapid detection of bacterial spores is essential to ensure food safety. Commercial availability of such a test will present a high potential for food sector. We present a point-of-need colorimetric assay for detection of Bacillus cytotoxicus spores in food. The detection principle is based on spore-enhanced peroxidase-like catalytic activity of gold nanoparticles. The sensing platform consists of a microtube containing gold nanoparticles (AuNPs), and magnetic particles (MPs), both conjugated with specific aptamer BAS6R that recognize B. cytotoxicus spores. Upon the addition of the sample, spores were determined as present by the enhanced color change of the solution, due to the oxidation of tetramethylbenidine (TMB) with H2O2. The assay was evaluated by the naked eye (on/off) and quantitatively with use of a spectrophotometer. BAS6R@AuNPs aptasensor coupled to BAS6R@MPs proved to be highly sensitive, achieving the naked-eye limit of detection as low as 102 cfu/mL in water and milk, and 104 cfu/mL in mashed potatoes. Moreover, discrimination between spores of B. cytotoxicus and B. subtilis as well as bacterial vegetative cells was achieved in contaminated food samples, providing a good selectivity. This work provides a promising proof of concept for the development of instrument-free, low-cost and rapid assay for Bacillus cytotoxicus spore detection, which is able to compete in sensitivity with conventional costly and time-consuming laboratory analyses.

Keywords

• Enzyme-free
• Colorimetry
• Gold nanoparticles
• Magnetic particles
• B. cereus spores
• Milk