Selective and Controllable Trapping of Single Proteins in Nanopores Using Reversible Covalent Bonds

Area of Science:

• Biophysics
• Nanotechnology
• Analytical Chemistry

Background:

• Nanopore analysis offers label-free, rapid determination of protein size and shape.
• Limitations include short protein residence times, high noise, and limited bandwidth, reducing analysis accuracy.
• Nonspecific protein interactions with nanopore walls further complicate accurate measurements.

Purpose of the Study:

• To develop a novel surface modification for solid-state nanopores to enhance single protein characterization.
• To selectively trap and analyze glycated proteins using functionalized nanopores.
• To improve the accuracy and throughput of nanopore-based protein size and shape determination.

Main Methods:

• Coating solid-state nanopores with a polymer and covalently attaching phenylboronic acid (PBA) groups.
• Utilizing the reversible covalent bonding between PBA and vicinal diols on glycated proteins for selective trapping.
• Analyzing resistive pulse dwell times to differentiate between free translocation and targeted trapping events.

Main Results:

• The functionalized nanopores selectively trap glycated proteins via PBA interactions, creating "long events" (0.4 ms to 2 s).
• Applied potential and buffer pH allow control over protein trapping times (1-2 orders of magnitude).
• Achieved highest accuracy to date for protein volume and shape determination using solid-state nanopores, with optimal trapping at 1-20 ms.

Conclusions:

• Polymer-coated, PBA-functionalized nanopores significantly improve selectivity and accuracy for single protein analysis.
• Controlled trapping extends protein residence time, enabling precise volume and shape determination.
• This method enhances nanopore analysis for both natively and artificially glycated proteins.