Impedance nanobiosensor based on enzyme-conjugated biosynthesized gold nanoparticles for the detection of Gram-positive bacteria

In this report, gold nanoparticles (GNPS) were synthesized using cell-free extracts of seven different isolates, namely, Pseudomonas aerogenosa CEBP2, Pseudomonas sp. CEBP1, Pseudomonas pseudoalcaligenes CEB1G, Acinetobactor baumani CEBS1, Cuprividus sp. CEB3, Micrococcus luteus CUB12, and Pandoraea sp. CUB2S. The spectroscopic (UV–vis, FTIR, DLS, XRD, EDS) and microscopic (FESEM, TEM) results confirm the reduction of Au³⁺ to Au⁰ in the presence of biomolecules having reducing as well as self-stabilizing activity. In this green synthesis approach, the average particle size of biosynthesized GNPS might vary (4–60 nm) depending on the bacterial species, pH of the media, incubation time, and temperature. In this study, GSH-modified BSGNPs (Au-GSH) have shown antimicrobial activity with better stability against Gram-positive bacteria. After conjugation of lysozyme with Au-GSH (lyso@Au-GSH), the zone of inhibition was enhanced from 12 to 23 mm (Au-GSH). The TEM study shows the spherical GNP (16.65 ± 2.84) turns into a flower-shaped GNP (22.22 ± 3.12) after conjugation with lysozyme due to the formation of the protein corona. Furthermore, the nanobioconjugate (lyso@Au-GSH) was immobilized with Nafion on a glassy carbon electrode to fabricate a label-free impedance biosensor that is highly sensitive to monitor changes in the transducer surface due to biomolecular interactions. The uniquely designed biosensor could selectively detect Gram-positive bacteria in the linear range of 3.0 × 10¹–3 × 10¹⁰ cfu mL⁻¹ with RE <5%. The proposed simplest biosensor exhibited good reproducibility (RSD = 3.1%) and excellent correlation (R² = 0.999) with the standard plate count method, making it suitable for monitoring Gram-positive bacterial contamination in biofluids, food, and environmental samples.