In‐situ PLL‐g‐PEG Functionalized Nanopore for Enhancing Protein Characterization

Single‐molecule nanopore detection technology has revolutionized proteomics research by enabling highly sensitive and label‐free detection of individual proteins. Herein, we designed a small, portable, and leak‐free flowcell made of PMMA for nanopore experiments. In addition, we developed an in situ functionalizing PLL‐g‐PEG approach to produce non‐sticky nanopores for measuring the volume of diseases‐relevant biomarker, such as the Alpha‐1 antitrypsin (AAT) protein. The in situ functionalization method allows continuous monitoring, ensuring adequate functionalization, which can be directly used for translocation experiments. The functionalized nanopores exhibit improved characteristics, including an increased nanopore lifetime and enhanced translocation events of the AAT proteins. Furthermore, we demonstrated the reduction in the translocation event's dwell time, along with an increase in current blockade amplitudes and translocation numbers under different voltage stimuli. The study also successfully measures the single AAT protein volume (253 nm3), which closely aligns with the previously reported hydrodynamic volume. The real‐time in situ PLL‐g‐PEG functionalizing method and the developed nanopore flowcell hold great promise for various nanopores applications involving non‐sticky single‐molecule characterization. In‐situ functionalization of thin PLL‐g‐PEG on SiN nanopores based on the self‐assembly and electrostatic interaction between the nanopore and PLL backbone of PLL‐g‐PEG is reported. This functionalization of SiN nanopores leads to non‐sticky protein translocation and volume estimation of individual proteins. Most significantly, our in situ method is monitorable for direct evaluation of functionalization before non‐sticky protein translocation.