Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore

Abstract

Nanopore analysis of nucleic acid is now routine, but detection of proteins remains challenging. Here, we report the systematic characterization of the effect of macromolecular crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). We observe a remarkable ca. 1000-fold increase in the molecule count for the globular protein β-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. We also demonstrate that macromolecular crowding facilitates the study of the topology of DNA plasmids and the characterization of amyloid fibril preparations with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addition of a macromolecular crowding agent to the electrolyte. We therefore envision that macromolecular crowding can be applied to many applications in the analysis of biomolecules by solid-state nanopores.

Sensing serotonin secreted from human serotonergic neurons using aptamer-modified nanopipettes

“…29 This phenomenon may be due to the capacity of nanopipettes to detect a small number of 5-HT molecules interacting with aptamers confined within the 10 nm orifice [47][48][49] or due to the macromolecular crowding effect in complex media that has been previously reported to increase detection sensitivity significantly. 50,51 What is more, the sensitivity of these 5-HT aptamer-modified nanopipettes that can detect concentrations of 5-HT in the low picomolar regime does not come at the cost of chemical selectivity. While other voltammetric techniques such as FSCV have existed for over five decades and have seen progress to advance rapid, sub-second neurotransmitter monitoring, 52 to date, the sensing surface (i.e.…”

Ribosome Fingerprinting with a Solid-State Nanopore

“…Further extension of this study to achieve a greater separation between consecutive ribosome numbers and increased sensitivity of the nanopore to fully map the polysomes is foreseeable in the future, for example, by better tuning the nanopore dimensions to the analyzed sample and by employing chemically functionalized nanopores. 39 Also, we envision that by taking advantage of the signal enhancement generated by macromolecular crowding 40 or by careful selection of the electrolyte, 41 we could better resolve the differences in dwell time and peak amplitude between the different polysome fractions. These improvements could pave the way for the use of solid-state nanopores to distinguish individual polysome fractions as an alternative or complement to sucrose density gradient ultracentrifugation but with the advantage of doing so at the single-entity level.…”

Ribosome fingerprinting with a solid-state nanopore

“…Further extension of this study to achieve a greater separation between consecutive ribosome numbers and increased sensitivity of the nanopore to fully map the polysomes is foreseeable in the future, for example by taking advantage of the signal enhancement generated by macromolecular crowding [36] or by careful selection of the electrolyte [37]. This could pave the way for the use of solid-state nanopores to distinguish individual polysome fractions as an alternative or complement to sucrose density gradient ultracentrifugation but with the advantage of doing so at the single entity level.…”