A miniaturized microscopy platform for plasmon-mediated single-molecule biosensing

Biosensing at the single-molecule level provides digital signals that enable the direct counting of individual molecular binding events. Single-molecule sensing provides digital signals, opening avenues toward kinetic fingerprinting while conferring higher tolerance to mechanical and temperature drift. Currently, single-molecule sensing is predominantly based on fluorescence probes. However, their weak signal poses challenges and necessitates the use of a research-grade microscope with a large footprint and high price. Here, we overcome these challenges and present a low-cost and compact microscopy platform for single-molecule biosensing. We quantify the performance of the platform where the use of a low numerical aperture lens results in a 10-fold reduced collection efficiency and a 6-fold increased background. Despite this, the strong digital signals provided by single-molecule plasmon-enhanced fluorescence are easily resolved, resulting in a limit of detection of 11 pM and a response time of 2 minutes. In addition, we demonstrate that the strong enhanced signals enable single-molecule detection of nucleic acid in diluted serum. Lastly, we integrate an automated pump and microfluidics to demonstrate continuous monitoring biosensing of a cancer marker on this miniaturized platform, thereby paving the way toward broad adoption of single-molecule devices for diagnostics.