Erik C. Yusko

Controlling protein translocation through nanopores with bio-inspired fluid walls

Synthetic nanopores have been used to study individual biomolecules in high thoroughput but their performance as sensors does not match biological ion channels. Controlling the translocation times of single-molecule analytes and their non-specific interaction with pore walls remain a challenge. Inspired by the olfactory sensilla of the insect antenna, here we show that coating nanopores with fluid bilayer lipids allows the pore diameters to be fine-tuned in sub-nanometre increments. Incorporation of mobile ligands in the lipid conferred specificity and slowed down the translocation of targeted proteins sufficiently to time-resolve translocation events of individual proteins. The lipid coatings also prevented pores from clogging, eliminated non-specific binding and enabled the translocation of amyloid-beta (Aβ) oligomers and fibrils. Through combined analysis of translocation time, volume, charge, shape and ligand affinity, different proteins were identified.

Applications of biological pores in nanomedicine, sensing, and nanoelectronics.

Biological protein pores and pore-forming peptides can generate a pathway for the flux of ions and other charged or polar molecules across cellular membranes. In nature, these nanopores have diverse and essential functions that range from maintaining cell homeostasis and participating in cell signaling to activating or killing cells. The combination of the nanoscale dimensions and sophisticated - often regulated - functionality of these biological pores make them particularly attractive for the growing field of nanobiotechnology. Applications range from single-molecule sensing to drug delivery and targeted killing of malignant cells. Potential future applications may include the use of nanopores for single strand DNA sequencing and for generating bio-inspired, and possibly, biocompatible visual detection systems and batteries. This article reviews the current state of applications of pore-forming peptides and proteins in nanomedicine, sensing, and nanoelectronics.

Electroosmotic flow can generate ion current rectification in nano- and micropores.

This paper introduces a strategy for generating ion current rectification through nano- and micropores. This method generates ion current rectification by electroosmotic-driven flow of liquids of varying viscosity (and hence varying conductance) into or out of the narrowest constriction of a pore. The magnitude of current rectification was described by a rectification factor, R(f), which is defined by the ratio of the current measured at a positive voltage divided by the current measured at a negative voltage. This method achieved rectification factors in the range of 5-15 using pores with diameters ranging from 10 nm to 2.2 microm. These R(f) values are similar to the rectification factors reported in other nanopore-based methods that did not employ segmented surface charges. Interestingly, this work showed that in cylindrical nanopores with diameters of 10 nm and a length of at least 275 nm, electroosmotic flow was present and could generate ion current rectification. Unlike previous methods for generating ion current rectification that require nanopores with diameters comparable to the Debye length, this work demonstrated ion current rectification in micropores with diameters 500 times larger than the Debye length. Thus this method extends the concept of fluidic diodes to the micropore range. Several experiments designed to alter or remove electroosmotic flow through the pore demonstrated that electroosmotic flow was required for the mode of ion current rectification reported here. Consequently, the magnitude of current rectification could be used to indicate the presence of electroosmotic flow and the breakdown of electroosmotic flow with decreasing ionic strength and hence increasing electric double layer overlap inside nanopores.

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.

Multivariate analyses of amyloid-beta oligomer populations indicate a connection between pore formation and cytotoxicity.

Aggregates of amyloid-beta (Aβ) peptides are thought to be involved in the development of Alzheimer’s disease because they can change synaptic plasticity and induce neuronal cell death by inflammation, oxidative damage, and transmembrane pore formation. Exactly which oligomeric species underlie these cytotoxic effects remains unclear. The work presented here established well-controlled aggregation conditions of Aβ 1–40 or Aβ1–42 peptides over a 20-day period and characterized these preparations with regard to their β-sheet content, degree of fibril formation, relative abundance of various oligomer sizes, and propensity to induce membrane pore formation and cytotoxicity. Using this multivariate data set, a systematic and inherently unbiased partial least squares (PLS) approach showed that for both peptides the abundance of oligomers in the tetramer to 13-mer range contributed positively to both pore formation and cytotoxicity, while monomers, dimers, trimers, and the largest oligomers (>210 kDa) were negatively correlated to both phenomena. Multivariate PLS analysis is ideally suited to handle complex data sets and interdependent variables such as relative oligomer concentrations, making it possible to elucidate structure function relationships in complex mixtures. This approach, therefore, introduces an enabling tool to the field of amyloid research, in which it is often difficult to interpret the activity of individual species within a complex mixture of bioactive species.

Single-Particle Characterization of Aβ 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-β oligomers directly in solution and without chemical modification. This method classified individual amyloid-β aggregates as spherical oligomers, protofibrils, or mature fibers and made it possible to account for the large heterogeneity of amyloid-β 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.

Ultrafast laser fabrication of submicrometer pores in borosilicate glass

We demonstrate rapid fabrication of submicrometer-diameter pores in borosilicate glass using femtosecond laser machining and subsequent wet-etch techniques. This approach allows direct and repeatable fabrication of high-quality pores with diameters of 400-800 nm. Such small pores coupled with the desirable electrical and chemical properties of glass enable sensitive resistive-pulse analysis to determine the size and concentration of macromolecules and nanoparticles. Plasma-enhanced chemical vapor deposition allows further reduction of pore diameters to below 300 nm.

Gramicidin Pores Report the Activity of Membrane-Active Enzymes

Phospholipases constitute a ubiquitous class of membrane-active enzymes that play a key role in cellular signaling, proliferation, and membrane trafficking. Aberrant phospholipase activity is implicated in a range of diseases including cancer, inflammation, and myocardial disease. Characterization of these enzymes is therefore important, both for improving the understanding of phospholipase catalysis and for accelerating pharmaceutical and biotechnological applications. This paper describes a novel approach to monitor, in situ and in real-time, the activity of phospholipase D (PLD) and phospholipase C (PLC) on planar lipid bilayers. This method is based on lipase-induced changes in the electrical charge of lipid bilayers and on the concomitant change in ion concentration near lipid membranes. The approach reports these changes in local ion concentration by a measurable change in the single channel ion conductance through pores of the ion channel-forming peptide gramicidin A. This enzyme assay takes advantage of the amplification characteristics of gramicidin pores to sense the activity of picomolar to nanomolar concentrations of membrane-active enzymes without requiring labeled substrates or products. The resulting method proceeds on lipid bilayers without the need for detergents, quantifies enzyme activity on native lipid substrates within minutes, and provides unique access to both leaflets of well-defined lipid bilayers; this method also makes it possible to generate planar lipid bilayers with transverse lipid asymmetry.

A Model for the Interfacial Kinetics of Phospholipase D Activity on Long-Chain Lipids

The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics.

Current oscillations generated by precipitate formation in the mixing zone between two solutions inside a nanopore

Unlike biological protein pores in lipid membranes, nanopores fabricated in synthetic materials can withstand a wide range of environmental conditions including the presence of organic solvents. This capability expands the potential of synthetic nanopores to monitor chemical reactions occurring at the interface between solutions of organic and aqueous character. In this work, nanopores fabricated in borosilicate glass or silicon nitride connected a predominantly organic solvent to an aqueous solvent, thereby generating a mixing zone between these solutions inside the pore. This configuration was exploited to precipitate small organic molecules with low aqueous solubility inside the nanopores, and concomitantly, to monitor this precipitation by the decrease of the ionic conductance through the nanopores over time. Hence, this method provides a means to induce and investigate the formation of nanoprecipitates or nanoparticles. Interestingly, precipitates with a slight electric charge were cleared from the pore, causing the conductance of the pore to return to its original value. This process repeated, resulting in stable oscillations of the ionic current. Although such oscillations might be useful in fluidic logic circuits, few conditions capable of generating oscillations in ionic currents have been reported. The frequency and amplitude of oscillations could be tuned by changing the concentration of the precipitating molecule, the pH of the electrolyte, and the applied potential bias. In addition to generating oscillations, nanopores that separate two different solutions may be useful for monitoring and mediating chemical reactions in the mixing zone between two solutions.