Exosome analysis is a promising tool for clinical and biological research applications. However, detection and biomarker quantification of exosomes is technically challenging because they are small and highly heterogeneous. Here...

Evanescent illumination has been widely used to detect single biological macromolecules because it can notably enhance light-analyte interaction. However, the current evanescent single-molecule detection system usually requires specially designed microspheres or nanomaterials. Here we show that single protein detection and imaging can be realized on a plain glass surface by imaging the interference between the evanescent lights scattered by the single proteins and by the natural roughness of the cover glass. This allows us to quantify the sizes of single proteins, characterize the protein–antibody interactions at the single-molecule level, and analyze the heterogeneity of single protein binding behaviors. In addition, owing to the exponential distribution of evanescent field intensity, the evanescent imaging system can track the analyte axial movement with high resolution, which can be used to analyze the DNA conformation changes, providing one solution for detecting small molecules, such as microRNA. This work demonstrates a label-free single protein imaging method with ordinary consumables and may pave a road for detecting small biological molecules.

Single-molecule detection can push beyond ensemble averages and reveal the statistical distributions of molecular properties. Measuring the binding kinetics of single proteins also represents one of the critical and challenging tasks in protein analysis. Here, we report total internal reflection-based evanescent scattering microscopy with label-free single-protein detection capability. Total internal reflection is employed to excite the evanescent field to enhance light− analyte interaction and reduce environmental noise. As a result, the system provides wide-field imaging capability and allows excitation and observation using one objective. In addition, this system quantifies protein binding kinetics by simultaneously counting the binding of individual molecules and recording their binding sites with nanometer precision, providing a digital method to measure binding kinetics with high spatiotemporal resolution. This approach does not employ specially designed microspheres or nanomaterials and may pave a way for label-free single-protein analysis in conventional microscopy.