Abstract
Rapid, sensitive, and portable pathogen detection is critical for infectious disease control and environmental public health surveillance but remains constrained by conventional methods requiring laborious sample pretreatment and bulky instrumentation. We rationally integrate immunosolid-phase microextraction (iSPME) with portable photothermal imaging (PI), enabling rapid, on-site pathogen detection in complex matrices. This platform combines two innovations: (1) antibody-functionalized SPME fibers with fibrous SiO2 microspheres (342.76 m2/g surface area) engineered via CTAB-templated synthesis, enabling rapid (15 min) and high-efficiency pathogen capture (73.7-98.9% recovery) through covalent antibody immobilization on carboxylated surfaces; (2) NIR-responsive plasmonic Aushell nanoprobes optimized via Ag/Au redox etching, achieving a red-shifted LSPR peak at 798 nm and a 57.1% photothermal conversion efficiency for interference-free signal transduction. The iSPME-PI platform eliminates sample transfer steps by forming a sandwich immunocomplex directly on the fiber, enabling spatially resolved photothermal quantification under an 808 nm laser excitation. It delivers ultralow detection limits (74.8 pg/mL SARS-CoV-2 N, 68.5 pg/mL Flu A NP) and robust performance in saliva, milk, and sewage (RSD < 8.3%). Clinical samples showed 100% concordance for SARS-CoV-2. Furthermore, wastewater monitoring using the iSPME-PI platform accurately tracked influenza A outbreaks in real time, demonstrating its potential for environmental biosurveillance and early warning systems.