Label-free Single-molecule Immunoassay

Single-molecule immunoassay is a reliable technique for the detection and quantification of low-abundance blood biomarkers, which are essential for early disease diagnosis and biomedical research. However, current single-molecule methods all require signal amplification via labelling, which brings a variety of unwanted consequences, such as matrix effects and autofluorescence interference. Here, we introduce a real-time mass imaging-based label-free single-molecule immunoassay (LFSM-immunoassay). Featuring plasmonic scattering microscopy-based real-time mass imaging, a 2-step sandwich assay format-enabled background reduction, and minimization of matrix effects by dynamic tracking of single binding events, the LFSM-immunoassay enables ultra-sensitive and direct protein detection at the single-molecule level in neat blood sample matrices. We demonstrated that the LFSM-immunoassay can measure sub-femtomolar levels of interleukin-6 and prostate-specific antigen in whole blood with 8 log dynamic range. To show its translational potential to clinical settings, we measured NT-proBNP (N-terminal pro-brain natriuretic peptide) in 28 patient serum samples using a 20 minute LFSM-immunoassay, and the results show a strong linear correlation (r > 0.99) with clinical lab reported values.