Probing the size of proteins with glass nanopores

Abstract

a Single molecule studies using nanopores have gained attention due to the ability to sense single molecules in aqueous solution without the need to label them. In this study, short DNA molecules and proteins were detected with glass nanopores, whose sensitivity was enhanced by electron reshaping which decreased the nanopore diameter and created geometries with a reduced sensing length. Further, proteins having molecular weights (MW) ranging from 12 kDa to 480 kDa were detected, which showed that their corresponding current peak amplitude changes according to their MW. In the case of the 12 kDa ComEA protein, its DNA-binding properties to an 800 bp long DNA molecule was investigated. Moreover, the influence of the pH on the charge of the protein was demonstrated by showing a change in the translocation direction. This work emphasizes the wide spectrum of detectable molecules using nanopores from glass nanocapillaries, which stand out because of their inexpensive, lithography-free, and rapid manufacturing process.

Selective single molecule nanopore sensing of proteins using DNA aptamer-functionalised gold nanoparticles

“…3,6 From these modulations one can extract information on molecular properties such as length, composition, and interactions with other biomolecules. 3,10 Although DNA based analysis has been most commonly performed, 11–13 more recently there has been a growing interest in protein based nanopore sensing to determine physical parameters such as size, 9,14 conformational state, 15,16 protein–protein interaction and binding kinetics between protein and antibodies/aptamers. 17–25 However, compared with DNA, proteins exhibit a diverse range of sizes, three-dimensional structures and have a non-uniform charge distribution, which introduces experimental challenges.…”

Beyond mass spectrometry, the next step in proteomics

“…Using the blockade current, nanopores larger than a protein have been used to detect and analyze native or folded proteins before (121)(122)(123)(124)(125)(126)(127)(128)(129)(130)(131)(132)(133)(134). Of particular interest are recent measurements using a wild-type aerolysin nanopore 1 to 1.7 nm in diameter through a lipid layer that have shown that it is possible to discriminate between several short, folded, uniformly charged, homopeptides and even identify single-AA differences (133).…”

Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor

“…For instance, synthetic nanopores for studying folding and unfolding of proteins, [ 6,[25][26][27][28][29][30][31][32] detecting protein analytes, [33][34][35] protein binding to nucleic acids, [36][37][38] and sizing of proteins and peptides. [39][40][41][42][43] Similarly, several biological pores, such as α-hemolysin, ClyA, and aerolysin have been employed for studying peptide fi brillation, [44][45][46] unraveling protein and peptide folding, [47][48][49][50][51][52][53][54][55][56] studying kinetics of protein binding, [ 57 ] as well as using aptamers for peptide sensing. [ 58,59 ] Solid-state pores generally can be fabricated with various pore sizes and geometry, but generating pores with reproducible thickness and dimensions with sub-nanometer precision is challenging.…”