Laser-made nanoparticle alternatives and machine learning-based image analysis for enhancing lateral flow immunoassay detection of bacterial β-lactamases

Gold nanoparticles (Au NPs) are widely used in colorimetric biosensing due to their unique plasmonic properties, producing a red color in traditional lateral flow immunoassays (LFIAs). However, their chemical synthesis is costly, and the global gold supply is increasingly limited. Additionally, visual detection often struggles to capture weak signals, leading to inconclusive results in point-of-care testing. In this study, we present a green, ligand-free method to synthesize gold (Au), platinum (Pt), and platinum–gold alloy (Pt–Au) nanoparticles using femtosecond laser ablation of metal targets in pure water—eliminating the need for chemical synthesis. This scalable, cost-effective approach enables the production of nanoparticles tailored for colorimetric LFIA and biosensing platforms. Pt and Pt–Au alloy NPs appear darker and, due to their catalytic properties, enhance signal contrast. We evaluated the sensing performance of these laser-synthesized NPs against commercial, chemically synthesized Au NPs using CMY-34 β-lactamase, a clinically relevant antibiotic resistance marker. Machine learning-assisted image analysis confirmed a 1 ng/ML limit of detection (LoD) for all three types of laser-synthesized NPs, surpassing traditional Au NPs. Furthermore, Pt–Au–based LFIA demonstrated improved detection accuracy over Au-only assays, highlighting the analytical advantages of bimetallic nanosystems. This study introduces an unbiased, automated LFIA image analysis pipeline and a sustainable NP synthesis method, including alloyed compositions, representing a major step forward in nanoparticle development for biosensing. Overall, this work paves the way for high-performance, cost-effective diagnostic technologies, expanding access to reliable biosensing in diverse healthcare and environmental settings.