3D-Photothermal Ultrafast PCR via rGO/PDMS-Based Microfluidic Chip and Its Application in Rapid Identification of Gram-Negative Bacteria in Whole Blood

Although polymerase chain reaction (PCR)-based molecular sensing methods offer faster and more accurate identification of bloodstream infection (BSI) pathogens compared to blood culture methods, conventional PCR platforms still face significant limitations, including bulky instrumentation, high energy consumption, and relatively long assay duration. Photothermal PCR enables accelerated thermal cycling and lower energy input; however, it often suffers from nonuniform heat distribution during ultrashort heating intervals, fluorescence quenching induced by nanomaterials, nonspecific adsorption of biomolecules, and complex device fabrication. To address these challenges, this study presents a novel microfluidic PCR system based on a patterned reduced graphene oxide/polydimethylsiloxane (rGO/PDMS) composite that enables three-dimensional photothermal heating. Beyond its simplified fabrication process, the platform demonstrates uniform heat transfer and eliminates nonspecific adsorption during amplification. Gram-negative bacteria constitute a substantial proportion of pathogens responsible for BSI. To further streamline bacterial detection from whole blood, a novel Gram-negative bacterial DNA extraction method employing antibiotic-modified magnetic beads was integrated into the workflow. The entire detection process, from sample preparation to result readout, can be completed within 40 min, with 40 PCR cycles accomplished in just 5 min. The system achieves a limit of detection for bacteria as low as 10² CFU mL^-1 in whole blood.