Real-time shape approximation and fingerprinting of single proteins using a nanopore.

Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.

Ion selectivity of graphene nanopores.

As population growth continues to outpace development of water infrastructure in many countries, desalination (the removal of salts from seawater) at high energy efficiency will likely become a vital source of fresh water. Due to its atomic thinness combined with its mechanical strength, porous graphene may be particularly well-suited for electrodialysis desalination, in which ions are removed under an electric field via ion-selective pores. Here, we show that single graphene nanopores preferentially permit the passage of K(+) cations over Cl(-) anions with selectivity ratios of over 100 and conduct monovalent cations up to 5 times more rapidly than divalent cations. Surprisingly, the observed K(+)/Cl(-) selectivity persists in pores even as large as about 20 nm in diameter, suggesting that high throughput, highly selective graphene electrodialysis membranes can be fabricated without the need for subnanometer control over pore size.

Single-particle characterization of Aß oligomers in solution.

Determining the pathological role of amyloids in amyloid-associated diseases will require a method for characterizing the dynamic distributions in size and shape of amyloid oligomers with high resolution. Here, we explored the potential of resistive-pulse sensing through lipid bilayer-coated nanopores to measure the size of individual amyloid-ß oligomers directly in solution and without chemical modification. This method classified individual amyloid-ß aggregates as spherical oligomers, protofibrils, or mature fibers and made it possible to account for the large heterogeneity of amyloid-ß aggregate sizes. The approach revealed the distribution of protofibrillar lengths (12- to 155 -mer) as well as the average cross-sectional area of protofibrils and fibers.

DNA characterization with ion beam-sculpted silicon nitride nanopores.

Solid-state nanopores are emerging as robust single molecule electronic measurement devices and as platforms for confining biomolecules for further analysis. The first silicon nitride nanopore to detect individual DNA molecules was fabricated using ion beam sculpting (IBS), a method that uses broad, low-energy ion beams to create nanopores with dimensions ranging from 2 to 20 nm. In this chapter, we discuss the fabrication, characterization, and use of IBS-sculpted nanopores as well as efficient uses of pClamp and MATLAB software suites for data acquisition and analysis. The fabrication section covers the repeatability and the pore size limits. The characterization discussion focuses on the geometric properties as measured by low- and high-resolution transmission electron microscopy (TEM), electron energy loss spectroscopy, and energy-filtered TEM. The section on translocation experiments focuses on how to use tools commonly available to the nanopore experimenter to determine whether a pore will be useful for experimentation or if it should be abandoned. A memory-efficient method of taking data using Clampex's event-driven mode and dual-channel recording is presented, followed by an easy-to-implement multithreshold event detection and classification method using MATLAB software.

Controlling protein translocation through nanopores with bio-inspired fluid walls.

Synthetic nanopores have been used to study individual biomolecules in high throughput, but their performance as sensors does not match that of biological ion channels. Challenges include control of nanopore diameters and surface chemistry, modification of the translocation times of single-molecule analytes through nanopores, and prevention of non-specific interactions with pore walls. Here, inspired by the olfactory sensilla of insect antennae, we show that coating nanopores with a fluid lipid bilayer tailors their surface chemistry and allows fine-tuning and dynamic variation of pore diameters in subnanometre increments. Incorporation of mobile ligands in the lipid bilayer conferred specificity and slowed the translocation of targeted proteins sufficiently to time-resolve translocation events of individual proteins. Lipid coatings also prevented pores from clogging, eliminated non-specific binding and enabled the translocation of amyloid-beta (Aß) oligomers and fibrils. Through combined analysis of their translocation time, volume, charge, shape and ligand affinity, different proteins were identified.