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<p>Techniques to analyze proteins often involves complex workflows and/or sophisticated equipment with modest limits-of-detection. While fluorescence spectroscopy can interrogate single molecules, it often requires fluorescence labeling with lasers and microscopes. We report herein a label-free approach for analyzing intact proteins using resistive pulse sensing (RPS). RPS data were secured using a unique RPS device, which we call a dual in-plane nanopore sensor, fabricated in a thermoplastic. The nanopore sensor was produced via nano-injection molding with critical structures of 30&#xa0;nm, enabling the detection of individual protein molecules and providing an approach toward their identification. Following nano-injection molding, the pore size could be reduced to ∼ 10&#xa0;nm using thermal fusion bonding of a cover plate to the molded substrate. The device architecture contained two in-plane nanopores flanking a nanochannel (50 × 50&#xa0;nm width × depth and 5&#xa0;µm length) that facilitated the measurement of the apparent electrophoretic mobilities of protein molecules in a label free manner via their molecular-dependent time-of-flight (ToF; time-difference between two consecutive RPS events—peak pair). We investigated four model proteins and collected multiple characteristics including RPS peak amplitude and dwell time, as well as an RPS-independent value, which was the ToF. Furthermore, we analyzed the temporal profiles of RPS events revealing distinct peak shapes for spherical and non-spherical proteins that were influenced by their rotational motion when resident within the nanopore.</p>

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Insights on using plastic-based dual in-plane nanopore sensors for differentiation and shape determinations of single protein molecules

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月21日

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  • Suresh Shivanka,
  • Farhad Shiri,
  • Maximillian Chibuike,
  • Collin McKinney,
  • Matthew Verber,
  • Junseo Choi,
  • Sunggook Park,
  • Adam R. Hall,
  • Steven A. Soper

摘要

Techniques to analyze proteins often involves complex workflows and/or sophisticated equipment with modest limits-of-detection. While fluorescence spectroscopy can interrogate single molecules, it often requires fluorescence labeling with lasers and microscopes. We report herein a label-free approach for analyzing intact proteins using resistive pulse sensing (RPS). RPS data were secured using a unique RPS device, which we call a dual in-plane nanopore sensor, fabricated in a thermoplastic. The nanopore sensor was produced via nano-injection molding with critical structures of 30 nm, enabling the detection of individual protein molecules and providing an approach toward their identification. Following nano-injection molding, the pore size could be reduced to ∼ 10 nm using thermal fusion bonding of a cover plate to the molded substrate. The device architecture contained two in-plane nanopores flanking a nanochannel (50 × 50 nm width × depth and 5 µm length) that facilitated the measurement of the apparent electrophoretic mobilities of protein molecules in a label free manner via their molecular-dependent time-of-flight (ToF; time-difference between two consecutive RPS events—peak pair). We investigated four model proteins and collected multiple characteristics including RPS peak amplitude and dwell time, as well as an RPS-independent value, which was the ToF. Furthermore, we analyzed the temporal profiles of RPS events revealing distinct peak shapes for spherical and non-spherical proteins that were influenced by their rotational motion when resident within the nanopore.