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.

Large-scale production of polyimide micropore-based flow cells for detecting nano-sized particles in fluids.

In diagnostic and sequencing applications, solid-state nanopores hold significant promise as a single-molecule sensing platform. The fabrication of precisely sized pores has traditionally been challenging, laborious, expensive, and inefficient, which has limited its applications until recently. To overcome this problem, this paper proposes a novel, reliable, cost-effective, portable, mass-productive, robust, and ease-of-use micropore flow cell that works based on the resistive pulse sensor (RPS) technique in order to distinguish the different sizes of c nanoparticles. RPS is a robust and informative technique that can provide valuable details of the size, shape, charge, and individual particle concentrations in the media. By femtosecond laser drilling of a polyimide substrate as an alternate material, translocation of 100, 300, and 350 nm polystyrene nanoparticles in PBS buffer was distinguished by 0.1, 1, and 2 nA current blockade levels, respectively. This is the first time a micropore has been opened in a polyimide membrane using a femtosecond laser in a single step. The experimental and theoretical investigation, scanning electron microscopy and focused ion beam spectroscopy were performed to comprehensively explain the micropore's performance. We showed that our innovative micropore-based flow cell could distinguish nano-sized particles in fluids, and it can be used in large-scale production because of its simplicity and cost-effectiveness.