Full‐Length Single Protein Molecules Tracking and Counting in Thin Silicon Channels

• DOI: 10.1002/adma.202314319
• Publication Date: 2024-03-10T04:39:45Z
• Authors: Shilo Ohayon; Liran Taib; Navneet Chandra V...; Marzia Iarossi; Ivy Bhattacharya; Barak Marom; Diana Huttner; Amit Meller
• Keywords: Vascular Endothelial Growth Factor A; Silicon; 0303 health sciences; 03 medical and health sciences; Humans; Proteins; Single Molecule Imaging

AbstractEmerging single‐molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra‐fast full‐length protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms, and low throughputs. Here, a single‐molecule method for parallel protein separation and tracking is introduced, yielding multi‐dimensional molecular properties used for their identification. Proteins are tagged by chemo‐selective dual amino‐acid specific labels and are electrophoretically separated by their mass/charge in custom‐designed thin silicon channel with subwavelength height. This approach allows analysis of thousands of individual proteins within a few minutes by tracking their motion during the migration. The power of the method is demonstrated by quantifying a cytokine panel for host‐response discrimination between viral and bacterial infections. Moreover, it is shown that two clinically‐relevant splice isoforms of Vascular endothelial growth factor (VEGF) can be accurately quantified from human serum samples. Being non‐destructive and compatible with full‐length intact proteins, this method opens up ways for antibody‐free single‐protein molecule quantification.