Dual-Modal Localized Surface Plasmon Resonance Biosensor for Rapid Bacterial Gram-Typing and Species Identification in Bronchoalveolar Lavage Fluid

Rapid pathogen identification is critical for timely antibiotic therapy in patients with pneumonia, yet current methods remain too slow or complex for routine clinical use. Here, we describe a localized surface plasmon resonance (LSPR)-based biosensor platform that detects Gram-positive and Gram-negative bacteria directly in bronchoalveolar lavage fluid (BALF) without nucleic acid extraction or amplification. Gold nano-islands (AuNIs) functionalized with antibodies against lipoteichoic acid (LTA) or lipopolysaccharide (LPS) enabled selective bacterial capture. The sensor achieved a refractive index resolution of 8.52 × 10-8 RIU with good reusability and dynamic range. Detection limits reached the concentration of 0.027 μM for purified LTA and 0.208 endotoxin units (EU) for LPS. When tested with Escherichia coli in artificial samples, the LPS sensor showed a reproducibility standard deviation (RSD) of 3.14%, detecting bacterial growth significantly earlier than conventional OD600 measurement. In 33 clinical BALF samples, sensor results correlated strongly with standard diagnostics (Pearson r = 0.963) and distinguished infected from noninfected cases using cutoff values of 0.060 rad (Gram-positive) and 0.083 rad (Gram-negative). The platform detects polymyxin B susceptibility 8 h earlier than conventional OD600 measurements, and a proof-of-concept sandwich immunoassay enables species-level discrimination between E. coli and Klebsiella pneumoniae. With excellent reproducibility (RSD = 3.14%), reusability over six cycles (84.21% ± 2.03% signal retention), and four-week storage stability (98.63% ± 5.42%), this LSPR platform offers a practical, rapid approach for frontline bacterial screening and accelerated antibiotic stewardship in pneumonia.