A microscale soft ionic power source modulates neuronal network activity.

Bio-integrated devices need power sources to operate1,2. Despite widely used technologies that can provide power to large-scale targets, such as wired energy supplies from batteries or wireless energy transduction3, a need to efficiently stimulate cells and tissues on the microscale is still pressing. The ideal miniaturized power source should be biocompatible, mechanically flexible and able to generate an ionic current for biological stimulation, instead of using electron flow as in conventional electronic devices4-6. One approach is to use soft power sources inspired by the electrical eel7,8; however, power sources that combine the required capabilities have not yet been produced, because it is challenging to obtain miniaturized units that both conserve contained energy before usage and are easily triggered to produce an energy output. Here we develop a miniaturized soft power source by depositing lipid-supported networks of nanolitre hydrogel droplets that use internal ion gradients to generate energy. Compared to the original eel-inspired design7, our approach can shrink the volume of a power unit by more than 105-fold and it can store energy for longer than 24 h, enabling operation on-demand with a 680-fold greater power density of about 1,300 W m-3. Our droplet device can serve as a biocompatible and biological ionic current source to modulate neuronal network activity in three-dimensional neural microtissues and in ex vivo mouse brain slices. Ultimately, our soft microscale ionotronic device might be integrated into living organisms.

Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate.

In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-molecule detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (~10(7) to 10(8) M(-1) · s(-1)), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.

Probing the orientational distribution of dyes in membranes through multiphoton microscopy.

Numerous dyes are available or under development for probing the structural and functional properties of biological membranes. Exogenous chromophores adopt a range of orientations when bound to membranes, which have a drastic effect on their biophysical behavior. Here, we present a method that employs optical anisotropy data from three polarization-imaging techniques to establish the distribution of orientations adopted by molecules in monolayers and bilayers. The resulting probability density functions, which contain the preferred molecular tilt µ and distribution breadth γ, are more informative than an average tilt angle [φ]. We describe a methodology for the extraction of anisotropy data through an image-processing technology that decreases the error in polarization measurements by about a factor of four. We use this technique to compare di-4-ANEPPS and di-8-ANEPPS, both dipolar dyes, using data from polarized 1-photon, 2-photon fluorescence and second-harmonic generation imaging. We find that di-8-ANEPPS has a lower tilt but the same distributional width. We find the distribution of tilts taken by di-4-ANEPPS in two phospholipid membrane models: giant unilamellar vesicles and water-in-oil droplet monolayers. Both models result in similar distribution functions with average tilts of 52° and 47°, respectively.

Protein detection by nanopores equipped with aptamers.

Protein nanopores have been used as stochastic sensors for the detection of analytes that range from small molecules to proteins. In this approach, individual analyte molecules modulate the ionic current flowing through a single nanopore. Here, a new type of stochastic sensor based on an αHL pore modified with an aptamer is described. The aptamer is bound to the pore by hybridization to an oligonucleotide that is attached covalently through a disulfide bond to a single cysteine residue near a mouth of the pore. We show that the binding of thrombin to a 15-mer DNA aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. The approach allows the quantification of nanomolar concentrations of thrombin, and provides association and dissociation rate constants and equilibrium dissociation constants for thrombin·aptamer interactions. Aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes.

Controlled translocation of individual DNA molecules through protein nanopores with engineered molecular brakes.

Protein nanopores may provide a cheap and fast technology to sequence individual DNA molecules. However, the electrophoretic translocation of ssDNA molecules through protein nanopores has been too rapid for base identification. Here, we show that the translocation of DNA molecules through the α-hemolysin protein nanopore can be slowed controllably by introducing positive charges into the lumen of the pore by site directed mutagenesis. Although the residual ionic current during DNA translocation is insufficient for direct base identification, we propose that the engineered pores might be used to slow down DNA in hybrid systems, for example, in combination with solid-state nanopores.

Transmembrane protein rotaxanes reveal kinetic traps in the refolding of translocated substrates.

Understanding protein folding under conditions similar to those found in vivo remains challenging. Folding occurs mainly vectorially as a polypeptide emerges from the ribosome or from a membrane translocon. Protein folding during membrane translocation is particularly difficult to study. Here, we describe a single-molecule method to characterize the folded state of individual proteins after membrane translocation, by monitoring the ionic current passing through the pore. We tag both N and C termini of a model protein, thioredoxin, with biotinylated oligonucleotides. Under an electric potential, one of the oligonucleotides is pulled through a α-hemolysin nanopore driving the unfolding and translocation of the protein. We trap the protein in the nanopore as a rotaxane-like complex using streptavidin stoppers. The protein is subjected to cycles of unfolding-translocation-refolding switching the voltage polarity. We find that the refolding pathway after translocation is slower than in bulk solution due to the existence of kinetic traps.

Multi-compartment encapsulation of communicating droplets and droplet networks in hydrogel as a model for artificial cells.

Constructing a cell mimic is a major challenge posed by synthetic biologists. Efforts to this end have been primarily focused on lipid- and polymer-encapsulated containers, liposomes and polymersomes, respectively. Here, we introduce a multi-compartment, nested system comprising aqueous droplets stabilized in an oil/lipid mixture, all encapsulated in hydrogel. Functional capabilities (electrical and chemical communication) were imparted by protein nanopores spanning the lipid bilayer formed at the interface of the encapsulated aqueous droplets and the encasing hydrogel. Crucially, the compartmentalization enabled the formation of two adjoining lipid bilayers in a controlled manner, a requirement for the realization of a functional protocell or prototissue.

Light-activated communication in synthetic tissues.

We have previously used three-dimensional (3D) printing to prepare tissue-like materials in which picoliter aqueous compartments are separated by lipid bilayers. These printed droplets are elaborated into synthetic cells by using a tightly regulated in vitro transcription/translation system. A light-activated DNA promoter has been developed that can be used to turn on the expression of any gene within the synthetic cells. We used light activation to express protein pores in 3D-printed patterns within synthetic tissues. The pores are incorporated into specific bilayer interfaces and thereby mediate rapid, directional electrical communication between subsets of cells. Accordingly, we have developed a functional mimic of neuronal transmission that can be controlled in a precise way.

Properties of Bacillus cereus hemolysin II: a heptameric transmembrane pore.

The gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(DeltaCT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell-free in vitro transcription and translation system, and were shown to assemble into SDS-stable oligomers on rabbit erythrocyte membranes and liposomes. The hemolytic activity of HlyII was measured with rabbit erythrocytes yielding an HC(50) value of 1.64 ng mL(-1), which is over 15 times more potent than staphylococcal alpha-hemolysin. HlyII(DeltaCT) was about eight times less potent than HlyII in this assay. Limited proteolysis of the oligomers formed by HlyII and HlyII(DeltaCT) on red cell membranes showed that the C-terminal extension is sensitive to digestion, while HlyII(DeltaCT) is protease resistant and migrates with an electrophoretic mobility similar to that of digested HlyII. HlyII forms moderately anion selective, rectifying pores (I(+80)/I(-80) = 0.57, 1 M KCl, pH 7.4) in planar lipid bilayers of diphytanoylphosphatidylcholine with a unitary conductance of 637 pS (1 M KCl, 5 mM HEPES, pH 7.4) and exhibits no gating over a wide range of applied potentials (-160 to +160 mV). In addition, it was demonstrated that HlyII forms a homoheptameric pore by using gel shift electrophoresis aided by a genetically encoded oligoaspartate tag. Although they share limited primary sequence identity (30%), these data confirm that HlyII is a structural and functional homolog of staphylococcal alpha-hemolysin.

Droplet interface bilayers.

Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.

Stochastic detection of enantiomers.

The rapid quantification of the enantiomers of small chiral molecules is very important, notably in pharmacology. Here, we show that the enantiomers of drug molecules can be distinguished by stochastic sensing, a single-molecule detection technique. The sensing element is an engineered alpha-hemolysin protein pore, fitted with a beta-cyclodextrin adapter. By using the approach, the enantiomeric composition of samples of ibuprofen and thalidomide can be determined in less than 1 s.

Direct introduction of single protein channels and pores into lipid bilayers.

We have developed a mechanical method for inserting single pores and channels into lipid bilayers. A hand-operated hydrogel probe, coated with a layer of proteins, is mechanically engaged with the lipid bilayer. The two major classes of membrane proteins (beta barrels and alpha-helix bundles) that can be inserted, thereby demonstrating the wide applicability of the approach. Recordings from the proteins show that they retain electrical properties that are the same as those of proteins inserted from solution. Protein-loaded probes can be used repeatedly, allowing individual pores to be rapidly screened one at a time. The method has implications for fundamental studies of cell membranes, array fabrication, and chemical screening.

Probing distance and electrical potential within a protein pore with tethered DNA.

DNA molecules tethered inside a protein pore can be used as a tool to probe distance and electrical potential. The approach and its limitations were tested with alpha-hemolysin, a pore of known structure. A single oligonucleotide was attached to an engineered cysteine to allow the binding of complementary DNA strands inside the wide internal cavity of the extramembranous domain of the pore. The reversible binding of individual oligonucleotides produced transient current blockades in single channel current recordings. To probe the internal structure of the pore, oligonucleotides with 5' overhangs of deoxyadenosines and deoxythymidines up to nine bases in length were used. The characteristics of the blockades produced by the oligonucleotides indicated that single-stranded overhangs of increasing length first approach and then thread into the transmembrane beta-barrel. The distance from the point at which the DNA was attached and the internal entrance to the barrel is 43 A, consistent with the lengths of the DNA probes and the signals produced by them. In addition, the tethered DNAs were used to probe the electrical potential within the protein pore. Binding events of oligonucleotides with an overhang of five bases or more, which threaded into the beta-barrel, exhibited shorter residence times at higher applied potentials. This finding is consistent with the idea that the main potential drop is across the alpha-hemolysin transmembrane beta-barrel, rather than the entire length of the lumen of the pore. It therefore explains why the kinetics and thermodynamics of formation of short duplexes within the extramembranous cavity of the pore are similar to those measured in solution, and bolsters the idea that a "DNA nanopore" provides a useful means for examining duplex formation at the single molecule level.

Partitioning of individual flexible polymers into a nanoscopic protein pore.

Polymer dynamics are of fundamental importance in materials science, biotechnology, and medicine. However, very little is known about the kinetics of partitioning of flexible polymer molecules into pores of nanometer dimensions. We employed electrical recording to probe the partitioning of single poly(ethylene glycol) (PEG) molecules, at concentrations near the dilute regime, into the transmembrane beta-barrel of individual protein pores formed from staphylococcal alpha-hemolysin (alphaHL). The interactions of the alpha-hemolysin pore with the PEGs (M(w) 940-6000 Da) fell into two classes: short-duration events (tau approximately 20 micro s), approximately 85% of the total, and long-duration events (tau approximately 100 micro s), approximately 15% of the total. The association rate constants (k(on)) for both classes of events were strongly dependent on polymer mass, and values of k(on) ranged over two orders of magnitude. By contrast, the dissociation rate constants (k(off)) exhibited a weak dependence on mass, suggesting that the polymer chains are largely compacted before they enter the pore, and do not decompact to a significant extent before they exit. The values of k(on) and k(off) were used to determine partition coefficients (Pi) for the PEGs between the bulk aqueous phase and the pore lumen. The low values of Pi are in keeping with a negligible interaction between the PEG chains and the interior surface of the pore, which is independent of ionic strength. For the long events, values of Pi decrease exponentially with polymer mass, according to the scaling law of Daoud and de Gennes. For PEG molecules larger than approximately 5 kDa, Pi reached a limiting value suggesting that these PEG chains cannot fit entirely into the beta-barrel.