Kinetic and equilibrium analysis of electrochemical Aptasensing for real‐time detection of <i>Staphylococcus aureus</i> in food substances

Real-time detection of foodborne pathogens such as Staphylococcus aureus (S. aureus) is essential for ensuring food safety. In this study, we evaluate the performance of an electrochemical aptasensor developed from gold nanoparticles (AuNPs)-immobilized screen-printed carbon electrode for the detection of low concentrations of S. aureus in chicken extract media. Using cyclic voltammetry (CV), the dynamic interaction between the aptamer-modified electrode and S. aureus was monitored across four bacterial concentrations of 1, 5, 10, and 20 colony-forming units per milliliter (CFU/mL) at 35-min intervals over 350 min. The aptasensor demonstrated a concentration-dependent response with increasingly lower maximum CV signals and faster time to equilibrium as CFU increased. Real-time kinetic and equilibrium parameters were extracted to understand the binding behavior of the pathogen to the electrode surface. Critical parameters such as the kinetic rate constant (k) of 0.0274 min^-1 and equilibrium dissociation constants ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>K</mml:mi> <mml:mi>d</mml:mi></mml:msub> </mml:mrow> </mml:math> ) of 7.35 CFU/mL, were derived from the CV signals. Langmuir isotherm modeling yielded a maximum binding capacity ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>B</mml:mi> <mml:mi>max</mml:mi></mml:msub> </mml:mrow> </mml:math> ) of 33.55 μA. In addition, a Hill coefficient (n_H) of 0.65 was obtained, which indicates a slightly negative cooperativity. These findings demonstrate the capability of the aptasensor for real-time detection of S. aureus, offering a robust framework for field-deployable pathogen monitoring in food matrices.