Addressing Buffer, Size, and Clogging Challenges in LAMP-Coupled Solid-State Nanopores for Point-of-Care Testing

Loop-mediated isothermal amplification (LAMP) is a promising method for point-of-care nucleic acid testing due to its simplicity, rapidity, and high sensitivity. Coupling LAMP with solid-state nanopores enables label-free, single-molecule sensing, enhancing diagnostic accuracy. However, conventional LAMP-coupled nanopore protocols require high-salt buffers (>1 M) to improve signal strength and translocation frequency, complicating workflows and increasing contamination risks. In native LAMP buffers (50 mM KCl), electroosmotic flow (EOF) hinders amplicon transport in sub-10 nm pores, while large amplicons increase the risk of clogging. These challenges limit event rates, data throughput, and device reliability. To address these limitations, we developed a glass nanopore device optimized for direct sensing of amplicons in native buffers, featuring integrated declogging capabilities. Our results revealed that 200 nm pores provided the best balance between minimizing EOF interference and maintaining strong signal strength, achieving the highest event rates. Smaller pores (<100 nm) had low event rates due to EOF effects, while larger pores (>1 μm) showed weakened signal strength. We discovered that clogging in low-salt conditions differs from high-salt environments, with physical vibration effectively resolving clogging in low-salt settings. This led to the integration of an automated vibration motor, extending nanopore lifespan and ensuring continuous data acquisition. Our clog-free, native-buffer sensing platform demonstrated a sensitivity of 0.12 parasite/μL using <i><i>Plasmodium vivax</i></i> (<i><i>P. vivax</i></i>) as a model organism, exceeding the threshold for detecting asymptomatic infections. These advancements highlight the potential of our nanopore device for rapid, reliable, and user-friendly diagnostics for point-of-care testing.