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

Abstract 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.