Optical Nanobubble Amplification Enables Compartment- and Enzyme-Free Digital Attomolar Protein Absolute Quantification

Absolute quantification at the single-protein level is crucial for accurately defining relationships between biomarkers, which are pivotal for disease diagnostics and therapeutic development. While digital detection technologies have achieved single-molecule sensitivity, they often rely on enzymatic amplification and physical compartmentalization, making the process complex and time-consuming. To address these challenges, we developed BEACON (biomarker enumeration via amplified cavitation optical nanobubbles), a compartment- and enzyme-free, high-throughput platform for single-protein counting. BEACON employs antibody-functionalized silica beads and gold nanoparticles to form sandwich complexes with the target proteins. Upon laser excitation, each gold nanoparticle generates a detectable plasmonic nanobubble, providing a robust scattering signal for the digital counting of individual proteins. Using BEACON, we achieved attomolar detection limits for clinically relevant protein biomarkers: PSA (32 aM), COVID-S (123 aM), and RSV fusion (65 aM). BEACON offers a simplified, rapid, and highly sensitive alternative to existing digital immunoassays with significant potential for clinical diagnostics and point-of-care applications.