Novel High‐Throughput Electrochemical Detection of <i>Staphylococcus Aureus</i>, <i>Bacillus Cereus</i>, or <i>Micrococcus Luteus</i> Using AuNPs@Ti₃C₂T_z Functionalized with Sandwich Peptides

Affinity-based electrochemical biosensors hold promise for detecting pathogenic bacteria in environmental applications. This study focuses on detecting gram-positive bacteria, which can cause fatal infections and are a major global mortality factor. An electrochemical biosensor platform using high-throughput 16-channel gold disk electrodes (16-GDEs) inspired by bio-microelectromechanical systems (BioMEMS) is developed, it incorporates a nanocomposite (AuNPs@Ti₃C₂T_z) with sandwich peptide structures to enhance electroconductivity and biological antifouling. Using AuNPs@Ti₃C₂T_z-coated 16-GDEs, sensitive biosensors for gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, or Micrococcus luteus) are constructed and validated in fresh-water samples by spiking with bacteria, which showed linear correlations between normalized peak current and logarithmic concentrations of the target bacteria (adjusted R-square ≥ 0.93). A single high-throughput platform containing biosensors for S. aureus, M. luteus, or B. cereus is also developed, exhibiting specific responses without any cross-reactivity in real samples. This platform enabled sensitive simultaneous detection of multiple analytes in environmental samples (500 CFU mL⁻¹) and can be applied to detect any target analyte with a suitable peptide pair. The strategy is to implement a quantitative real-time polymerase chain reaction (RT-qPCR) adaptive sensing device to successfully detect gram-positive bacteria. The nanocomposite-enabled electrochemical biosensor platform on 16-GDEs offers a valuable tool for environmental and clinical diagnostics.