Synthesis and Characterization of Thermostable Antimicrobial Peptide–DNAzyme Conjugates for the Proof-of-Concept Detection of E. coli

Bacterial infections are a common cause of morbidity and mortality worldwide. The early detection of pathogens is crucial to minimize health risks, control spread, and prevent outbreaks. Current detection methods are either cultivation-based and time-consuming or rely on sophisticated, costly assays that require specialist equipment, making them unsuitable for rapid and cost-efficient screening, e.g., in food safety. Here, we investigated antimicrobial peptides (AMPs) conjugated to HRP-mimicking DNAzymes as a potentially novel class of broad-spectrum bacterial biosensors. AMPs bind to bacterial cells, and the DNAzyme catalyzes a peroxidase reaction exploitable for sensing applications. We established and optimized a synthesis protocol to prepare AMP-DNAzyme conjugates, synthesized several AMP/DNAzyme combinations, and characterized them. Molecular dynamics simulations of selected conjugates were conducted to gain insight into their binding capacity. We also investigated the potential of these constructs for the proof-of-concept detection of a clinicalEscherichia coliisolate on two low-cost detection platforms: a syringe microfilter system and an ELISA-like assay. On the syringe microfilter, we detected 108 cells/mL within 30 min, while the ELISA-like assay achieved a limit of detection below 5 cells/mL, resulting in 1-2 orders of magnitude improved sensitivity compared to similar AMP-based biosensors. This highly stable and cost-efficient biosensor can have potential applications in fields such as environmental monitoring, food safety, and quality control, where a broad approach is used to detect the presence of bacterial contamination.