Label-free digital protein sensing by integral plasmonic imaging

The capability to detect trace-level protein in complex biofluids is critical in various fields, from fundamental biological research to clinical disease diagnosis. Digital protein sensing by counting single molecules bound to probes is the technology of choice with exceptional sensitivity. However, label-free single-molecule sensing has been challenging so far because of the extremely weak signal and inherent quantum noise. Herein, we present an integral plasmonic imaging technique for the next generation of label-free digital protein sensing, offering in-plane plasmonic scattering and integral detection to maximize optical scattering signals and collection efficiency. This technology allows for high-sensitivity single-molecule detection, without the need for signal amplification, and provides the capability to simultaneously obtain mass and binding kinetics information. Leveraging the plasmonic waveguide mode, the system ensures low light-induced heating effects, making it highly suitable for biological applications. Experimental results demonstrate quantitative imaging and digital counting of individual unlabeled protein molecules, including representative globular proteins down to 25 kDa, and enable detection of protein concentrations at the sub-picomolar level in buffered solutions and biologically relevant fluid environments. Additionally, the system’s unique ability to perform digital counting of molecular binding events highlights its potential for in-depth analysis of binding kinetics. We anticipate that this label-free digital protein sensing could find wide applications in clinical low-abundance molecular detection. Ultrasensitive and specific protein analysis is critical to early diagnosis of diseases and fundamental biological research. Direct optical detection of mass and binding at the single-protein level provides a promising route to such analysis, but it has been challenging due to shot noise imposed by the quantum nature of light. We find that the integral plasmonic imaging scheme could break the above fundamental limit. It enables single-protein analysis with reduced illumination intensity and extended bandwidth while maintaining state-of-the-art sensitivity. This work paves the way towards label-free digital protein analysis and advanced optical measurement techniques.