ナノポアにおける可逆的共有結合を用いた単一タンパク質の選択的かつ制御可能な捕捉

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.