Single protein molecules separation, tracking and counting in ultra-thin silicon channels

Emerging single-molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra-fast full-size protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms and low throughputs. Here, we introduce a single-molecule method for parallel protein separation and tracking, yielding multi-dimensional molecular properties used for their identification. Proteins are tagged by dual amino-acid specific labels and are electrophoretically separated by their mass/charge in custom-designed silicon nano-channel. This approach allows us to analyze thousands of individual proteins within a few minutes by tracking their motion during the migration. We demonstrate the power of the method by quantifying a cytokine panel for host-response discrimination between viral and bacterial infections. Moreover, we show that two clinically-relevant splice isoforms of VEGF can be accurately quantified from human serum samples. Being non-destructive and compatible with full-length intact proteins, this method opens up new ways for antibody-free single protein molecule quantification.