Label-Free Dynamic Single-Molecule Sensing of Alpha-2-Macroglobulin in Complex Matrices

Alpha-2-macroglobulin (A2M) is a critical biomarker implicated in inflammation, immune regulation, coagulation, and various pathological conditions such as liver fibrosis, neurodegenerative diseases, and cancers. However, its precise quantification remains challenging due to complex conformational dynamics, subtle abundance fluctuations, and interference from plasma proteins. Here, we present a label-free dynamic single-molecule sensing (LFDSMS) strategy for the sensitive and specific detection of A2M. By monitoring reversible interactions between A2M and surface-immobilized antibodies in real time, and optimizing buffer ionic strength to accelerate binding-dissociation kinetics, this method achieves efficient time-dependent signal amplification and robust detection performance. The platform achieves limits of detection of 25 ± 2 ng/mL in undiluted serum and 29 ± 4 ng/mL in cell culture medium, while maintaining high specificity in complex biological matrices. Furthermore, LFDSMS shows excellent agreement with conventional ELISA results (<i>R</i>² = 0.988), validating its potential for quantitative biomarker profiling in clinically relevant samples.