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

## TL;DR

This paper introduces a method to selectively trap and analyze individual proteins using nanopores with reversible covalent bonds, improving accuracy and selectivity.

## Contribution

A novel polymer coating with phenylboronic acid groups enables selective trapping of glycated proteins in nanopores via reversible covalent bonds.

## Key findings

- Two populations of resistive pulses were observed: short pulses from free translocation and long events from covalent trapping.
- Controlling applied potential or pH extends trapping times by one to two orders of magnitude, improving analysis accuracy.
- Trapping times of 1 to 20 ms are optimal for reliable volume and shape analysis of proteins.

## Abstract

Analysis of individual proteins using nanopores makes
it possible
to determine their size and shape in a label-free approach, within
minutes, and from μL sample volumes. Short residence times of
proteins in the nanopore, high electrical current noise, and bandwidth
limitations of the recording electronics during resistive pulse recordings,
however, limit the accuracy of size and shape analysis of individual
proteins. The work presented here introduces a polymer surface coating
of solid-state nanopores to minimize nonspecific interactions of proteins
with the nanopore wall while functionalizing it covalently with phenylboronic
acid (PBA) groups. These PBA groups make it possible to trap selectively
glycated proteins by taking advantage of the formation of reversible
covalent bonds between PBA and vicinal diol groups of glycated amino
acid residues on proteins. Dwell time analysis revealed two populations
of resistive pulses: short pulses with dwell times t
d below 0.4 ms from free translocation of proteins and
resistive pulses that we term “long events” that last
from 0.4 ms to 2 s and result from intended transient covalent bonds
between glycated proteins and PBA groups in the nanopore lumen. The
choice of applied potential differences during nanopore recordings
or the pH value of the recording buffer makes it possible to control
and extend the most probable trapping time of proteins in the nanopore
within one to 2 orders of magnitude. This approach provides the highest
accuracy for the determination of protein volume and shape achieved
to date with solid-state nanopores and reveals that a trapping time
of 1 to 20 ms is ideal to achieve reliable volume and shape analysis
while retaining high throughput of the analysis. This approach, hence,
extends the residence time of natively glycated proteins or of proteins
that are intentionally glycated by straightforward incubation in a
glucose solution, thereby providing selectivity and improving the
accuracy of nanopore-based characterization of single proteins.