In silico design of magnetic, polymeric synthetic receptor targeting clumping factor A, for the specific capture and...

Rapid diagnosis of Staphylococcus aureus (S. aureus) is critical for both therapy and infection control programs. Currently available rapid bacterial detection methods such as polymerase chain reaction (PCR) requires expensive equipment and trained personnel, whereas, enzyme-linked immunosorbent assay (ELISA) requires antibodies and thus, suffer from limitations such as limited reagent stability. Herein, we used stable, cost-effective alternates to the antibodies known as synthetic antibodies i.e., molecularly imprinted polymers (MIPs). In this study, polymeric synthetic receptor commonly known as MIPs were layered onto magnetic nanoparticles, specifically designed for the detection of S. aureus through the binding interaction with its surface biomarker- clumping factor A (ClfA). This approach offers a low limit of detection (LOD) of 10² colony-forming units per mL (CFU/mL) and a wide linear detection range (10³ to 10⁸ CFU/mL) for S. aureus. Briefly, ClfA gene was cloned, expressed and protein was purified using Ni-NTA affinity chromatography and anion-exchange chromatography. Magnetic nanoparticles were initially synthesized and coated with silica, followed by introduction of aldehyde groups for immobilization through imine bonding. ClfA was then immobilized onto the functionalized nanoparticles, serving as a template for MIP synthesis. To determine a monomer combination with high binding capacity and specificity for ClfA, docking studies were performed using Autodock 4.2. The polymerization process employed selected monomer combination, yielding MIP tailored to recognize ClfA. The binding properties of the MIP were extensively investigated, demonstrating specificity and selectivity for ClfA over non-specific proteins. Furthermore, the clinical utility of the MIP was assessed by examining its binding with ClfA in serum samples. The present study contributes to the advancement of specific and efficient tools for the S. aureus diagnostics, based on a virulence biomarker, ClfA, emphasizing the potential applications of molecularly imprinted magnetic nanoparticles for the detection of microorganisms and their virulence.