A novel smartphone-based nanozyme-enhanced electrochemical immunosensor for ultrasensitive direct detection of Staphylococcus aureus in milk and blood serum

Abstract

A graphene quantum dot (GQD)-based nanozyme-enhanced electrochemical sensor was developed for the ultra-sensitive and rapid Staphylococcus aureus detection in PBS, undiluted milk, and blood serum. The S. aureus concentration for immunosensor calibration was electrochemically determined to be 1.24× 10⁹ CFU mL⁻¹ (R² = 0.99), utilizing an innovative smartphone-based electrochemical device. Transmission electron (TEM) and atomic force (AFM) microscopy analyses of GQDs revealed uniform nanoparticles (8–10 nm) with surface heights of 2 nm and 21 nm at different scan areas. Fluorescence and Fourier transform infrared spectroscopy spectra showed an emission at 530 nm with an excitation wavelength of 485 nm, along with the presence of hydroxyl, carboxyl, and aromatic groups on the GQD surfaces. The maximum current peak for GQDs was obtained by optimizing electrochemical properties through amperometry at +0.9 V. The optimal signal intensity- and limit of detection (LOD)-based GQD concentrations were 0.0125, 0.5, and 1 mg mL⁻¹ for PBS, undiluted milk, and blood serum, respectively. The optimal antibody concentration for S. aureus detection was 25 μg mL⁻¹, resulting in a 91 % amperometric suppression and a 94 % fluorescence quenching. The developed biosensor effectively detected S. aureus, with calculated LODs of 1, 4, and 344 CFU mL⁻¹ in PBS, undiluted milk, and blood serum, respectively. TEM, AFM, and contact angle analyses confirmed S. aureus binding to the GQD-antibody bioconjugate, shifting surface height to 35 nm and contact angle from ~30° to ~65°. The immunosensor showed high specificity for S. aureus with minimal cross-reactivity to Listeria monocytogenes, Staphylococcus epidermidis, Enterococcus faecium, and Streptococcus pneumoniae.