The routinely used enzymes, antibodies, and nucleic acids-based biosensors for detection of Staphylococcus aureus are often overwhelmed by limited selectivity, sensitivity, high cost, and inability to discriminate between live/dead cells. This necessitates the development of an ultra-sensitive, stable, and selective electrochemical biosensor capable of discriminating live S. aureus in a mixture of live/dead cells in food samples. The current study reports the development of an electrochemical biosensor through the immobilization of bacteriophage in surface-modified bacterial cellulose (BC) matrix. BC being highly porous and fibrous, offers a high surface area for the impregnation of carboxylated multiwalled carbon nanotubes (c-MWCNTs) and allows high-density phage immobilization. Surface modification of BC/c-MWCNTs with polyethyleneimine (PEI) provides a positive charge that facilitates oriented phage immobilization. FE-SEM and FT-IR analyses confirmed the development of BC/c-MWCNTs-PEI-phage bio-interface. Confocal microscopy analysis showed 11.7 ± 1.2 phage particles⋅μm-2 immobilized in the BC matrix and showed anti-staphylococcal activity by producing clear lytic zone and reduced bacterial growth. Differential pulse voltammetry (DPV) analysis detected 3 CFU⋅mL-1 and 5 CFU⋅mL-1 of S. aureus in phosphate buffer saline (PBS) and milk, respectively, within 30 min at neutral pH and showed stability over 6-weeks at 4 °C. The biosensor showed high specificity for S. aureus, both in pure and mixed cultures of non-host bacteria, and effectively discriminated live S. aureus in a mixture of live/dead cells. The developed biosensor represents a simple, sensitive, specific, and accurate tool for early detection of S. aureus in food samples.
Keywords: Bacterial cellulose; Electrochemical biosensor; High-density phage immobilization; Live/dead cell discrimination; Sensitivity and selectivity; Staphylococcus aureus detection.
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