Abstract
This study introduces a novel biosensing approach for the detection of pathogen DNA in breast milk utilizing single carbon fiber electrodes (SCFE) enhanced with MXene nanomaterial layers. The primary innovation lies in the modification of SCFE with MXenes to increase the electrode's surface area, followed by surface activation for the immobilization of dCas9-sgRNA complexes. This modification aims to leverage the unique properties of MXenes and the selective binding capability of the CRISPR technology for efficient and specific pathogen detection. Electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM) analyses were employed to characterize the electrode modifications and the immobilization process, demonstrating the successful enhancement of biosensor performance. This study further optimized the chronoimpedimetric detection method to achieve rapid, sensitive, and selective detection of Staphylococcus aureus (SAu) DNA in breast milk, with a notable detection time of 60 s in real samples. The biosensor demonstrated high selectivity and sensitivity, with a linear detection range between 50 and 6000 fM and a limit of detection (LOD) of 14.5 fM. The reproducibility and stability of the biosensor were also confirmed through multiple tests, showing promising potential for clinical and public health applications.