FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers

Abstract

Iron-regulated surface determinant protein A (IsdA) is a key surface protein found in the foodborne bacteriaStaphylococcus aureus (S. aureus)which is known to be critical for bacterial survival and colonization. S. aureus is pathogenic and has been linked to foodborne diseases; thus, early detection is critical to prevent diseases caused by this bacterium. Despite IsdA being a specific marker for S. aureus and several detection methods have been developed for sensitive detection of this bacteria such as cell culture, nucleic acids amplification, and other colorimetric and electrochemical methods, the detection of S. aureus through IsdA is underdeveloped. Here, by combining computational generation of target-guided aptamers and fluorescence resonance energy transfer (FRET)-based single-molecule analysis, we presented a widely applicable and robust detection method for IsdA. Three different RNA aptamers specific to the IsdA protein were identified and their ability to switch a FRET construct to a high-FRET state in the presence of protein was verified. The presented approach demonstrated the detection of IsdA down to picomolar levels (×10–12 M, equivalent to ∼1.1 femtomoles IsdA) with a dynamic range extending to ∼40 nM. The FRET-based single-molecule technique that we reported here is capable of detecting the foodborne pathogen protein IsdA with high sensitivity and specificity and has a broader application in the food industry and aptamer-based sensing field by enabling quantitative detection of a wide range of pathogen proteins.

Recent Advances in Aptamer-Based Biosensors for Bacterial Detection

“…For instance, RNA aptamers have been reported that target teichoic acid in S. aureus [53], a component of the cell wall of most Gram-positive bacteria, as well as peptidoglycan-specific aptamers for the detection of S. aureus and E. coli [54] and, more recently, aptamers targeting isdA, a crucial surface protein for S. aureus' survival and colonization. Over time, this protein has gradually become a key marker for the detection of this bacterium [55,56].…”

Aptamer-Based Biosensors for Bacterial Detection

“…More recently, aptamers that specifically target isdA, a crucial surface protein for S. aureus survival and colonization, have been successfully generated. Over time, this protein is gradually becoming a key marker for the detection of this bacterium [49,50].…”