Proteins are the structural elements and machinery of life responsible for a functioning biological architecture and homeostasis. Advances in nanotechnology are catalyzing key breakthroughs in many areas, including the analysis and study of proteins at the single-molecule level. Nanopore sensing is at the forefront of this revolution. This tutorial review provides readers a guidebook and reference for detecting and characterizing proteins at the single-molecule level using nanopores. Specifically, the review describes the key materials, nanoscale features, and design requirements of nanopores. It also discusses general design requirements as well as details on the analysis of protein translocation. Finally, the article provides the background necessary to understand current research trends and to encourage the identification of new biomedical applications for protein sensing using nanopores.

BackgroundEnteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored.ResultsWe utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally.ConclusionsOur data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0376-y) contains supplementary material, which is available to authorized users.

No abstract

Monitoring individual proteins in solution while simultaneously obtaining tertiary and quaternary structural information is challenging. In this study, translocation of the vascular endothelial growth factor (VEGF) protein through a solid-state nanopore (ssNP) produces distinct ion-current blockade amplitude levels and durations likely corresponding to monomer, dimer, and higher oligomeric states. Upon changing from a non-reducing to a reducing condition, ion-current blockage events from the monomeric state dominate, consistent with the expected reduction of the two inter-chain VEGF

No abstract

Self-assembly of amyloid nanofiber is associated with functional and pathological processes such as in neurodegenerative diseases. Despite intensive studies, stochastic nature of the process has made it difficult to elucidate molecular mechanisms for the key amyloid nucleation. Here, we investigated the amyloid nucleation of silk-elastinlike peptide (SELP) using time-lapse lateral force microscopy (LFM). By repeated scanning a single line on a SELP-coated mica surface, we observed sudden stepwise height increases, corresponds to nucleation of an amyloid fiber. The lateral force profiles followed either a worm-like chain model or an exponential function, suggesting that the atomic force microscopy (AFM) tip stretches a single or multiple SELP molecules along the scanning direction, serves as the template for further selfassembly perpendicular to the scan direction. Such mechanically induced nucleation of amyloid fibrils allows positional and directional control of amyloid assembly in vitro, which we demonstrate by generating single nanofibers at predetermined nucleation sites.

Solid-state nanopores are an emerging biosensor for nucleic acid and protein characterization. For use in a clinical setting, solid-state nanopore sensing requires sample preparation and purification, fluid handling, a heating element, electrical noise insulators, and an electrical readout detector, all of which hamper its translation to a point-of-care diagnostic device. A stand-alone microfluidicbased nanopore device is described that combines a bioassay reaction/purification chamber with a solid-state nanopore sensor. The microfluidic device is composed of the high-temperature/solvent resistance Zeonex plastic, formed via micro-machining and heat bonding, enabling the use of both a heat regulator and a magnetic controller. Fluid control through the microfluidic channels and chambers is controlled via fluid port selector valves and allows up-to eight different solutions. Electrical noise measurements and DNA translocation experiments demonstrate the integrity of the device, with performance comparable to a conventional stand-alone nanopore setup. However, the microfluidic-nanopore setup is superior in terms of ease of use. To showcase the utility of the device, single molecule detection of a DNA PCR product, after magnetic bead DNA separation, is accomplished on chip.

Immobilization of biosensors in or on a functional material is critical for subsequent device development and translation to wearable technology. Here, we present the development and assessment of an immobilized quantum dot− transcription factor−nucleic acid complex for progesterone detection as a first step toward such device integration. The sensor, composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA, is embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot−transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot−transcription factor DNA assembly resulting in an attenuated ratiometric fluorescence output via Forster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeated analyte measurements are similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor−DNA assembly as a recognition/transduction element, offering a promising framework for future biosensor devices.