Macrofluidic Device for Preparative Concentration Based on Epitachophoresis.

We have developed a new separation device to concentrate and collect ions from several milliliter sample volumes to microliter fractions. Unlike most conventional platforms, this device has circular architecture. The electrophoretic migration operates from the outer perimeter toward the center. Separations can be performed both in continuous (zone electrophoresis) and discontinuous (moving boundary) electrolyte systems. We use a discontinuous electrolyte system comprising a leading and a terminating electrolyte to concentrate samples containing small organic anions and DNA fragment. The agarose gel stabilizes the boundary between the leading and terminating electrolytes. The milliliter volume sample is mixed with the terminating electrolyte and migrates through the gel toward the center. The concentrated total sample is collected in microliter fraction at the center. The potential for preparative concentration of DNA is demonstrated using a DNA ladder. Because zone migration accelerates as it moves toward the center, we named this method Epitachophoresis from the Greek word "επιταχυνω (epitachýnο)", meaning "acceleration". To the best of our knowledge, this unique circular architecture has not been previously described.

Stochastic Detection of MPSA-Gold Nanoparticles Using a α-Hemolysin Nanopore Equipped with a Noncovalent Molecular Adaptor.

We present the first study of a novel, more sensitive method for the characterization of nanoparticles (NPs). This approach combines detection via a protein nanopore with modification of its interaction behavior using a molecular adaptor. We identify different populations of 3-mercapto-1-propanesulfonate (MPSA)-modified-gold NPs using the biological nanopores α-hemolysin (αHL) and its M113N mutant equipped with a noncovalently bound γ-cyclodextrin molecule as a stochastic sensor. Identification takes place on the basis of the extent of current blockades and residence times. Here, we demonstrate that noncovalently attached adaptors can be used to change the sensing properties of αHL nanopores, allowing the detection and characterization of different populations of MPSA NPs. This is an advance in sensitivity and diversity of NP sensing, as well as a promising and reliable technology to characterize NPs by using protein nanopores.

Sensing single mixed-monolayer protected gold nanoparticles by the α-hemolysin nanopore.

Gold nanoparticles are widely used in various applications in fields including chemistry, engineering, biology, medicine, and electronics. These materials can be synthesized and modified with ligands containing different functional groups. Among nanoparticles' characteristics, chemical surface composition is likely to be a crucial feature, demanding robust analytical methodologies for its assessment. Single molecule analysis using the biological nanopores α-hemolysin and its E111A mutant is presented here as a promising methodology to stochastically sense organic monolayer protected gold-nanoparticles with different ligand shell compositions. By monitoring the ionic current across a single protein nanopore, differences in the physical and chemical characteristics (e.g., size, ligand shell composition, and arrangement) of individual nanoparticles can be distinguished based on the differences in the current blockade events that they cause. Such differences are observed in the spread of both the amplitude and duration of current blockades. These values cannot be correlated with a single physical characteristic. Instead the spread represents a measure of heterogeneity within the nanoparticle population. While our results compare favorably with the more traditional analytical methodologies, further work will be required to improve the accuracy of identification of the NPs and understand the spread of values within a nanoparticle preparation as well as the overlap between similar preparations.

The role of Lys147 in the interaction between MPSA-gold nanoparticles and the α-hemolysin nanopore.

Single channel recordings were used to determine the effect of direct electrostatic interactions between sulfonate-coated gold nanoparticles and the constriction of the Staphylococcus aureus α-hemolysin protein channel on the ionic current amplitude. We provide evidence that Lys147 of α-hemolysin can interact with the sulfonate groups at the nanoparticle surface, and these interactions can reversibly block 100% of the residual ionic current. Lys147 is normally involved in a salt bridge with Glu111. The capture of a nanoparticle leads to a partial current block at neutral pH values, but protonation of Glu111 at pH 2.8 results in a full current block when the nanoparticle is captured. At pH 2.8, we suggest that Lys147 is free to engage in electrostatic interactions with sulfonates at the nanoparticle surface. To verify our results, we engineered a mutation in the α-hemolysin protein, where Glu111 is substituted by Ala (E111A), thus removing Glu111-Lys147 interactions and facilitating Lys147-sulfonate electrostatic interactions. This mutation leads to a 100% current block at pH 2.8 and a 92% block at pH 8.0, showing that electrostatic interactions are formed between the nanopore and the nanoparticle surface. Besides demonstrating the effect of electrostatic interactions on cross channel ionic current, this work offers a novel approach to controlling open and closed states of the α-hemolysin nanopore as a function of external gears.

3D printed device for epitachophoresis.

Polyacrylamide or agarose gels are the most frequently used sieving and stabilizing media in slab gel electrophoresis. Recently, we have introduced a new electrophoretic technique for concentration/separation of milliliter sample volumes. In this technique, the gel is used primarily as an anticonvection media eliminating liquid flow during the electromigration. While serving well for the liquid stabilization, the gels can undergo deformation when exposed to a discontinuous electrolyte buffer system used in epitachophoresis. In this work, we have explored 3D printing to form rigid stabilizing manifolds to minimize liquid flow during the epitachophoresis run. The whole device was printed using the stereolithography technique from a low water-absorbing resin. The stabilizing manifold, serving as the gel substitute, was printed as a replaceable composite structure preventing electrolyte mixing during the separation. Different geometries of the 3D printed stabilizing manifolds were tested for use in concentrating ionic sample components without spatial separation. The presented device can focus analytes from 3 or 4 mL of the sample to 150 µL or less, depending on the collection cup size. With the 150 µL collection cup, this represents the enrichment factor from 20 to 27. The time of concentration was from 15 to 25 min, depending on stabilization media and power used.

Epitachophoresis is a novel versatile total nucleic acid extraction method.

Epitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt-290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing.

Fabrication and practical applications of molybdenum disulfide nanopores.

Among the different developed solid-state nanopores, nanopores constructed in a monolayer of molybdenum disulfide (MoS2) stand out as powerful devices for single-molecule analysis or osmotic power generation. Because the ionic current through a nanopore is inversely proportional to the thickness of the pore, ultrathin membranes have the advantage of providing relatively high ionic currents at very small pore sizes. This increases the signal generated during translocation of biomolecules and improves the nanopores' efficiency when used for desalination or reverse electrodialysis applications. The atomic thickness of MoS2 nanopores approaches the inter-base distance of DNA, creating a potential candidate for DNA sequencing. In terms of geometry, MoS2 nanopores have a well-defined vertical profile due to their atomic thickness, which eliminates any unwanted effects associated with uneven pore profiles observed in other materials. This protocol details all the necessary procedures for the fabrication of solid-state devices. We discuss different methods for transfer of monolayer MoS2, different approaches for the creation of nanopores, their applicability in detecting DNA translocations and the analysis of translocation data through open-source programming packages. We present anticipated results through the application of our nanopores in DNA translocations and osmotic power generation. The procedure comprises four parts: fabrication of devices (2-3 d), transfer of MoS2 and cleaning procedure (24 h), the creation of nanopores within MoS2 (30 min) and performing DNA translocations (2-3 h). We anticipate that our protocol will enable large-scale manufacturing of single-molecule-analysis devices as well as next-generation DNA sequencing.

Gel-on-a-chip: continuous, velocity-dependent DNA separation using nanoscale lateral displacement.

We studied the trajectories of polymers being advected while diffusing in a pressure driven flow along a periodic pillar nanostructure known as nanoscale deterministic lateral displacement (nanoDLD) array. We found that polymers follow different trajectories depending on their length, flow velocity and pillar array geometry, demonstrating that nanoDLD devices can be used as a continuous polymer fractionation tool. As a model system, we used double-stranded DNA (dsDNA) with various contour lengths and demonstrated that dsDNA in the range of 100-10 000 base pairs (bp) can be separated with a size-selective resolution of 200 bp. In contrast to spherical colloids, a polymer elongates by shear flow and the angle of polymer trajectories with respect to the mean flow direction decreases as the mean flow velocity increases. We developed a phenomenological model that explains the qualitative dependence of the polymer trajectories on the gap size and on the flow velocity. Using this model, we found the optimal separation conditions for dsDNA of different sizes and demonstrated the separation and extraction of dsDNA fragments with over 75% recovery and 3-fold concentration. Importantly, this velocity dependence provides a means of fine-tuning the separation efficiency and resolution, independent of the nanoDLD pillar geometry.

Protein nanopores with covalently attached molecular adapters.

Molecular adapters are crucial for the stochastic sensing of organic analytes with alpha-hemolysin (alphaHL) protein nanopores when direct interactions between analytes and the pore cannot readily be arranged by conventional protein engineering. In our earlier studies, cyclodextrin adapters were lodged noncovalently within the lumen of the alphaHL pore. In the present work, we have realized the controlled covalent attachment of a beta-cyclodextrin (betaCD) adapter in the two possible molecular orientations inside alphaHL pores prepared by genetic engineering. There are two advantages to such a covalent system. First, the adapter cannot dissociate, which means there are no gaps during stochastic detection, a crucial advance for single-molecule exonuclease DNA sequencing where the continuous presence of a molecular adapter will be essential for reading individual nucleotides. Second, the ability to orient the adapter allows analytes to bind through only one of the two entrances to the betaCD cavity. We demonstrate that the covalently attached adapters can be used to alter the ion selectivity of the alphaHL pore, examine binding events at elevated temperatures, and detect analytes with prolonged dwell times.

Protein components for nanodevices.

A long-term goal of nanobiotechnology is to build tiny devices that respond to the environment, perform computations and carry out tasks. Considerable progress has been made in building protein components for such devices, and here we describe examples, including self-assembling protein arrays, pores with triggers and switches, and motor proteins harnessed for specific tasks. A major issue that has been successfully addressed in this recent work is the interface between the proteins and other components of the system, such as a metal surface. While further progress is expected in the coming years, the assembly of devices from the components has seen more limited accomplishments. For example, although a wide variety of sensors based on nanobiotechnology has been developed, unresolved problems still confront the construction of complex nanobioelectronic circuits, and the development of nanorobotics with biological components remains a distant dream.

Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20†nm.

Deterministic lateral displacement (DLD) pillar arrays are an efficient technology to sort, separate and enrich micrometre-scale particles, which include parasites, bacteria, blood cells and circulating tumour cells in blood. However, this technology has not been translated to the true nanoscale, where it could function on biocolloids, such as exosomes. Exosomes, a key target of 'liquid biopsies', are secreted by cells and contain nucleic acid and protein information about their originating tissue. One challenge in the study of exosome biology is to sort exosomes by size and surface markers. We use manufacturable silicon processes to produce nanoscale DLD (nano-DLD) arrays of uniform gap sizes ranging from 25 to 235 nm. We show that at low Péclet (Pe) numbers, at which diffusion and deterministic displacement compete, nano-DLD arrays separate particles between 20 to 110 nm based on size with sharp resolution. Further, we demonstrate the size-based displacement of exosomes, and so open up the potential for on-chip sorting and quantification of these important biocolloids.

Hydrodynamics of diamond-shaped gradient nanopillar arrays for effective DNA translocation into nanochannels.

Effective DNA translocation into nanochannels is critical for advancing genome mapping and future single-molecule DNA sequencing technologies. We present the design and hydrodynamic study of a diamond-shaped gradient pillar array connected to nanochannels for enhancing the success of DNA translocation events. Single-molecule fluorescence imaging is utilized to interrogate the hydrodynamic interactions of the DNA with this unique structure, evaluate key DNA translocation parameters, including speed, extension, and translocation time, and provide a detailed mapping of the translocation events in nanopillar arrays coupled with 10 and 50 µm long channels. Our analysis reveals the important roles of diamond-shaped nanopillars in guiding DNA into as small as 30 nm channels with minimized clogging, stretching DNA to nearly 100% of their dyed contour length, inducing location-specific straddling of DNA at nanopillar interfaces, and modulating DNA speeds by pillar geometries. Importantly, all critical features down to 30 nm wide nanochannels are defined using standard photolithography and fabrication processes, a feat aligned with the requirement of high-volume, low-cost production.

Fixed-gap tunnel junction for reading DNA nucleotides.

Previous measurements of the electronic conductance of DNA nucleotides or amino acids have used tunnel junctions in which the gap is mechanically adjusted, such as scanning tunneling microscopes or mechanically controllable break junctions. Fixed-junction devices have, at best, detected the passage of whole DNA molecules without yielding chemical information. Here, we report on a layered tunnel junction in which the tunnel gap is defined by a dielectric layer, deposited by atomic layer deposition. Reactive ion etching is used to drill a hole through the layers so that the tunnel junction can be exposed to molecules in solution. When the metal electrodes are functionalized with recognition molecules that capture DNA nucleotides via hydrogen bonds, the identities of the individual nucleotides are revealed by characteristic features of the fluctuating tunnel current associated with single-molecule binding events.

Fabrication of sub-20 nm nanopore arrays in membranes with embedded metal electrodes at wafer scales.

We introduce a method to fabricate solid-state nanopores with sub-20 nm diameter in membranes with embedded metal electrodes across a 200 mm wafer using CMOS compatible semiconductor processes. Multi-layer (metal-dielectric) structures embedded in membranes were demonstrated to have high uniformity (± 0.5 nm) across the wafer. Arrays of nanopores were fabricated with an average size of 18 ± 2 nm in diameter using a Reactive Ion Etching (RIE) method in lieu of TEM drilling. Shorts between the membrane-embedded metals were occasionally created after pore formation, but the RIE based pores had a much better yield (99%) of unshorted electrodes compared to TEM drilled pores (<10%). A double-stranded DNA of length 1 kbp was translocated through the multi-layer structure RIE-based nanopore demonstrating that the pores were open. The ionic current through the pore can be modulated with a gain of 3 using embedded electrodes functioning as a gate in 0.1 mM KCl aqueous solution. This fabrication approach can potentially pave the way to manufacturable nanopore arrays with the ability to electrically control the movement of single or double-stranded DNA inside the pore with embedded electrodes.

Electrical method to quantify nanoparticle interaction with lipid bilayers.

Understanding as well as rapidly screening the interaction of nanoparticles with cell membranes is of central importance for biological applications such as drug and gene delivery. Recently, we have shown that "striped" mixed-monolayer-coated gold nanoparticles spontaneously penetrate a variety of cell membranes through a passive pathway. Here, we report an electrical approach to screen and readily quantify the interaction between nanoparticles and bilayer lipid membranes. Membrane adsorption is monitored through the capacitive increase of suspended planar lipid membranes upon fusion with nanoparticles. We adopt a Langmuir isotherm model to characterize the adsorption of nanoparticles by bilayer lipid membranes and extract the partition coefficient, K, and the standard free energy gain by this spontaneous process, for a variety of sizes of cell-membrane-penetrating nanoparticles. We believe that the method presented here will be a useful qualitative and quantitative tool to determine nanoparticle interaction with lipid bilayers and consequently with cell membranes.

Nanoscale protein pores modified with PAMAM dendrimers.

We describe nanoscale protein pores modified with a single hyperbranched dendrimer molecule inside the channel lumen. Sulfhydryl-reactive polyamido amine (PAMAM) dendrimers of generations 2, 3 and 5 were synthesized, chemically characterized, and reacted with engineered cysteine residues in the transmembrane pore alpha-hemolysin. Successful coupling was monitored using an electrophoretic mobility shift assay. The results indicate that G2 and G3 but not G5 dendrimers permeated through the 2.9 nm cis entrance to couple inside the pore. The defined molecular weight cutoff for the passage of hyperbranched PAMAM polymers is in contrast to the less restricted accessibility of flexible linear poly(ethylene glycol) polymers of comparable hydrodynamic volume. Their higher compactness makes sulfhydryl-reactive PAMAM dendrimers promising research reagents to probe the structure of porous membrane proteins with wide internal diameters. The conductance properties of PAMAM-modified proteins pores were characterized with single-channel current recordings. A G3 dendrimer molecule in the channel lumen reduced the ionic current by 45%, indicating that the hyperbranched and positively charged polymer blocked the passage of ions through the pore. In line with expectations, a smaller and less dense G2 dendrimer led to a less pronounced current reduction of 25%. Comparisons to recordings of PEG-modified pores revealed striking dissimilarities, suggesting that differences in the structural dynamics of flexible linear polymers vs compact dendrimers can be observed at the single-molecule level. Current recordings also revealed that dendrimers functioned as ion-selectivity filters and molecular sieves for the controlled passage of molecules. The alteration of pore properties with charged and hyperbranched dendrimers is a new approach and might be extended to inorganic nanopores with applications in sensing and separation technology.

Stochastic detection of motor protein-RNA complexes by single-channel current recording.

A label- and immobilization-free approach to detecting the reversible formation of complexes between nucleic acids and proteins at the single-molecule level is described. The voltage-driven translocation of individual oligoribonucleotides through a nanoscale protein pore is observed by single-channel current recordings. The oligoribonucleotide 5'-C25A(25)-3' gives rise to current blockades with an average duration of approximately 0.5 ms. In the presence of the RNA-binding ATPase P4, a viral packaging motor from bacteriophage phi8, longer events of tens to hundreds of milliseconds are observed. Upon addition of ATP the long events disappear, indicating the dissociation of the P4RNA complex. The frequency of events also depends on the concentration of P4 and the length of the oligoribonucleotide, thereby confirming the specificity of the P4RNA events. This study shows that single-channel current recordings can be used to monitor RNA-protein complex formation, thus opening up a new means to examine the motor activity of RNA- or DNA-processing enzymes.

Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter.

Individual nucleic acid molecules might be sequenced by the identification of nucleoside 5'-monophosphates as they are released by processive exonucleases. Here, we show that single molecule detection with a modified protein nanopore can be used to identify ribonucleoside and 2'-deoxyribonucleoside 5'-monophosphates, thereby taking a step along this path. Distinct levels of current block are observed for each of the four members of a set of nucleoside 5'-monophosphates when the molecules bind within a mutant alpha-hemolysin pore, (M113R)(7), equipped with the molecular adapter heptakis-(6-deoxy-6-amino)-beta-cyclodextrin. While our results compare favorably with alternative approaches, further work will be required to improve the accuracy of identification of the nucleic acid bases, to feed each released nucleotide into the pore, and to ensure that every nucleotide is captured by the adapter.