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In-situ PLL-g-PEG Functionalized Nanopore for Enhancing Protein Characterization.

Mostafa Salehirozveh1,2, Anne-Kathrine Kure Larsen3,4,5, Milos Stojmenovic6

  • 1Department Of Physics And Astronomy, University of Bologna, Bologna, Italy.

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Summary

We developed a portable nanopore flowcell and an in-situ functionalization method for sensitive, label-free protein detection. This system accurately measures biomarker volume, advancing single-molecule analysis.

Keywords:
FlowcellIn situ functionalizationNanoporeNon-stickyProtein

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Area of Science:

  • Biophysics
  • Nanotechnology
  • Proteomics

Background:

  • Single-molecule nanopore detection offers sensitive, label-free protein analysis.
  • Existing methods face challenges with non-specific binding and nanopore stability.

Purpose of the Study:

  • To develop a portable, leak-free flowcell for nanopore experiments.
  • To create an in-situ functionalization method for non-sticky nanopores.
  • To measure the volume of disease biomarkers like Alpha-1 antitrypsin (AAT) protein.

Main Methods:

  • Designed a portable PMMA flowcell for nanopore experiments.
  • Developed an in-situ poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) functionalization approach.
  • Utilized nanopore translocation events to measure single AAT protein volume.

Main Results:

  • The functionalized nanopores showed increased lifetime and enhanced AAT protein translocation.
  • Reduced dwell time and increased current blockade amplitudes were observed under voltage.
  • Successfully measured single AAT protein volume (253 nm³), matching hydrodynamic volume predictions.

Conclusions:

  • The real-time in-situ functionalization method improves nanopore performance.
  • The developed flowcell and functionalization technique are promising for non-sticky single-molecule characterization.
  • This approach advances label-free biomarker quantification using nanopore technology.