Development of novel DNA aptamers and colorimetric nanozyme aptasensor for targeting multi-drug-resistant, invasive Salmonella typhimurium strain SMC25

Invasive, biofilm-forming Non-typhoidal Salmonella (iNTS), propagating through the global food and water supply chain, presents a significant risk to food safety and public health. Developing a robust detection system is crucial for enabling point-of-care, affordable, and equipment-free identification of this pathogen throughout the supply chain. In this study, we screened a novel pool of ssDNA aptamers specific to a multidrug resistant iNTS strain SMC25, previously isolated from Indian poultry products in our earlier research. Through 13 rounds of whole-cell SELEX, we identified, characterized, and selected seven full-length aptamers (ST18, ST19, ST25, ST28, ST29, ST31, and ST32). Flow cytometric analysis reveals superior binding of ST25, ST28, ST29, and ST31. These aptamers were translated onto Nanozyme-based aptasensing system for efficient, cost-effective detection of SMC25. This system harnesses the aptamer-mediated, reversible peroxidase-like activity of gold nanoparticles (GNPs) to oxidize the TMB substrate into a one-electron oxidation state, resulting in a blue-colored Diamine charge transfer complex (DCTC). The catalytic process, coupled with GNP aggregation, induces a visible color change in the test mixture from ruby-red to blue. Post-SELEX truncations identified the optimal aptamer sequence (T_ST31), which selectively detected SMC25 in water with a limit of detection (LOD) of ∼10⁴ CFU/mL. Lower concentrations (10 CFU/mL) of SMC25 could be detected after non-selective enrichment within 120 min. This research introduces a novel pool of iNTS-specific aptamers along with a cost-effective (0.25 USD per sample) solution for colorimetric detection by the naked eye. • Novel pool of ssDNA aptamers was identified against MDR, invasive Non-typhoidal Salmonella (iNTS) strain SMC25. • Cost-effective ($0.24 per sample) colorimetric nanozyme aptasensor was developed. • Post-SELEX truncations of the top-performing aptamers, ST28 and ST31, led to improved specificity. • Aptamer T_ST31 demonstrated a detection limit of approximately 10⁴ CFU/mL in non-enriched water samples. • Detection of iNTS strains in water was achieved within 2 h by enrichment in non-selective media like nutrient broth.