Quantitative Analysis of the Membrane Affinity of Local Anesthetics Using a Model Cell Membrane.

Local anesthesia is a drug that penetrates the nerve cell membrane and binds to the voltage gate sodium channel, inhibiting the membrane potential and neurotransmission. It is mainly used in clinical uses to address the pain of surgical procedures in the local area. Local anesthetics (LAs), however, can be incorporated into the membrane, reducing the thermal stability of the membrane as well as altering membrane properties such as fluidity, permeability, and lipid packing order. The effects of LAs on the membrane are not yet fully understood, despite a number of previous studies. In particular, it is necessary to analyze which is the more dominant factor, the membrane affinity or the structural perturbation of the membrane. To analyze the effects of LAs on the cell membrane and compare the results with those from model membranes, morphological analysis and 50% inhibitory concentration (IC50) measurement of CCD-1064sk (fibroblast, human skin) membranes were carried out for lidocaine (LDC) and tetracaine (TTC), the most popular LAs in clinical use. Furthermore, the membrane affinity of the LAs was quantitatively analyzed using a colorimetric polydiacetylene assay, where the color shift represents their distribution in the membrane. Further, to confirm the membrane affinity and structural effects of the membranes, we performed an electrophysiological study using a model protein (gramicidin A, gA) and measured the channel lifetime of the model protein on the free-standing lipid bilayer according to the concentration of each LA. Our results show that when LAs interact with cell membranes, membrane affinity is a more dominant factor than steric or conformational effects of the membrane.

Light-Responsive Polymeric Micellar Nanoparticles with Enhanced Formulation Stability.

Light-sensitive polymeric micelles have recently emerged as promising drug delivery systems for spatiotemporally controlled release of payload at target sites. Here, we developed diazonaphthoquinone (DNQ)-conjugated micellar nanoparticles that showed a change in polarity of the micellar core from hydrophobic to hydrophilic under UV light, releasing the encapsulated anti-cancer drug, doxetaxel (DTX). The micelles exhibited a low critical micelle concentration and high stability in the presence of bovine serum albumin (BSA) solution due to the hydrophobic and π-π stacking interactions in the micellar core. Cell studies showed enhanced cytotoxicity of DTX-loaded micellar nanoparticles upon irradiation. The enhanced stability would increase the circulation time of the micellar nanoparticles in blood, and enhance the therapeutic effectiveness for cancer therapy.

Employing LiCl salt gradient in the wild-type α-hemolysin nanopore to slow down DNA translocation and detect methylated cytosine.

In this research, we demonstrate a label-free detection, biological nanopore-based method to distinguish methylated cytosine (mC) from naked cytosine (C) in sample mixtures containing both C and mC at a prolonged translocation duration. Using a 15-fold increase in LiCl salt concentration going from a cis to trans chamber, we increased the translocation dwell time of ssDNA by over 5-fold and the event capture rate by 6-fold in comparison with the symmetric concentration of 1.0 M KCl (control). This is a consequence of counter-ion binding and effective lowering of the overall charge of DNA, which in turn lessens the electrophoretic drive of the system and slows the translocation velocity. Moreover, salt gradients can create a large electric field that will funnel ions and polymers towards the pore, increasing the capture rate and translocation dwell time of DNA. As a result, in 0.2 M-3.0 M LiCl solution, ssDNA achieved a prolonged dwell time of 52 µs per nucleotide and a capture rate of 60 ssDNA per second. Importantly, lowering the translocation speed of ssDNA enhances the resulting resolution, allowing 5'-mC to be distinguished from C without using methyl-specific labels. We successfully distinguished 5'-mC from C when mixed together at ratios of 1 : 1, 3 : 7 and 7 : 3. The distribution of current blockade amplitudes of all mixtures adopted bimodal shapes, with peak-to-peak ratios coarsely corresponding to the mixture composition (e.g. the density and distribution of events shifted in correspondence with changes in 18b-0mC and 18-2mC concentration ratios in the mixture).

Increased dwell time and occurrence of dsDNA translocation events through solid state nanopores by LiCl concentration gradients.

Solid-state nanopore based biosensors are cost effective, high-throughput engines for single molecule detection of biomolecules with the added benefit of size modification. Progress in the translation of the science into a viable diagnostic tool is impeded by inadequate sensitivity of data acquisition systems in detection of fast DNA translocations through the pore. To combat this, slowing the transport of DNA through the nanopore by use of various media or by altering experimental parameters is common. Applying a concentration gradient of KCl in the experimental ionic solution has been shown to effectively prolong dwell times as well as increase the capture rate of DNA by the nanopore. Our previous work has corroborated the ability of LiCl ionic solution to slow down the transport of dsDNA through the nanopore by up to 10-fold through cation-DNA interactions. However, this drastically reduced the event occurrence frequency, thus hindering the efficacy of this system as a reliable biosensor downstream. Here, we present the use of a concentration gradient of lithium chloride ionic solution to increase the event frequency of single molecule dsDNA translocation through a solid state nanopore. By using 0.5 M/3 M LiCl on the cis/trans chambers respectively, average dwell times experienced up to a 3-fold increase when compared to experiments run in symmetric 1 M LiCl. Additionally, experiments using the 0.5 M/3 M displayed a greater than 10-fold increase in event frequency, confirming the capture propensity of the asymmetric conditions.

Nanopore based detection of Bacillus thuringiensis HD-73 spores using aptamers and versatile DNA hairpins.

A versatile nanopore sensing platform to detect any aptamer using nanopores by designing DNA hairpins that are complementary to the aptamer is described. This platform can detect the presence of aptamer binding targets regardless of their size, which has been a major hurdle for nanopore detection systems. Moreover, the signal-to-noise ratio is increased by eliminating most of the unwanted substances from the sample via simple sample preparation steps. To detect Bacillus thuringiensis HD-73 spores using this sensing platform, DNA hairpins complementary to the target-specific aptamers were designed, and the hairpins were characterized using alpha-hemolysin nanopores after the reaction of spores and aptamers and subsequent reaction with the complementary DNA hairpins. The platform exhibited a detection limit as low as 1.2 × 101 CFU mL-1 and was compatible with a wide range of spore concentrations from 1.2 × 101 CFU mL-1 to 1.2 × 106 CFU mL-1 while it is still expandable to higher spore concentrations.

Solid-state nanopore fabrication in LiCl by controlled dielectric breakdown.

Nanopore fabrication via the controlled dielectric breakdown (CDB) method offers an opportunity to create solid-state nanopores directly in salt solution with sub-nanometer precision. Driven by trap assisted current tunneling, the method uses localized defects, or traps, in the dielectric material to isolate a breakdown point and fabricate a single pore in less than 10 minutes. Here we present an approach to controlled dielectric breakdown of SiNx in which the nanopore is fabricated in LiCl buffer instead of the traditional KCl buffer. Direct fabrication in LiCl buffer promotes a uniform, symmetric, cylindrical nanopore structure that is fully wet and can be used for experiments in situ. We have shown that fabrication in LiCl reduces the necessity for overnight pore stabilization and allows for the desired analyte to be added in significantly less time than it would take if fabrication was performed in KCl. Pores created by this approach can be used for biosensing applications, including the detection of double-stranded DNA. DNA translocation experiments were conducted in both LiCl and KCl buffer. Experiments conducted in LiCl buffer resulted in about a 2-fold increase in dsDNA transport duration when compared to experiments conducted in KCl buffer of the same concentration. An increase in transport duration of over 10-fold in comparison to KCl was observed when the concentration of the LiCl buffer was increased by a factor of 3.

Investigation of compacted DNA structures induced by Na+ and K+ monovalent cations using biological nanopores.

In aqueous solutions, an elongated, negatively charged DNA chain can quickly change its conformation into a compacted globule in the presence of positively charged molecules, or cations. This well-known process, called DNA compaction, is a method with great potential for gene therapy and delivery. Experimental conditions to induce these compacted DNA structures are often limited to the use of common compacting agents, such as cationic surfactants, polymers, and multivalent cations. In this study, we show that in highly concentrated buffers of 1 M monovalent cation solutions at pH 7.2 and 10, biological nanopores allow real-time sensing of individual compacted structures induced by K+ and Na+, the most abundant monovalent cations in human bodies. Herein, we studied the ratio between compacted and linear structures for 15-mer single-stranded DNA molecules containing only cytosine nucleotides, optimizing the probability of linear DNA chains being compacted. Since the binding affinity of each nucleotide to cation is different, the ability of the DNA strand to fold into a compacted structure greatly depends on the type of cations and nucleotides present. Our experimental results compare favorably with findings from previous molecular dynamics simulations for the DNA compacting potential of K+ and Na+ monovalent cations. We estimate that the majority of single-stranded DNA molecules in our experiment are compacted. From the current traces of nanopores, the ratio of compacted DNA to linear DNA molecules is approximately 30 : 1 and 15 : 1, at a pH of 7.2 and 10, respectively. Our comparative studies reveal that Na+ monovalent cations have a greater potential of compacting the 15C-ssDNA than K+ cations.

Detection of methylation on dsDNA using nanopores in a MoS2 membrane.

Methylation at the 5-carbon position of the cytosine nucleotide base in DNA has been shown to be a reliable diagnostic biomarker for carcinogenesis. Early detection of methylation and intervention could drastically increase the effectiveness of therapy and reduce the cancer mortality rate. Current methods for detecting methylation involve bisulfite genomic sequencing, which are cumbersome and demand a large sample size of bodily fluids to yield accurate results. Hence, more efficient and cost effective methods are desired. Based on our previous work, we present a novel nanopore-based assay using a nanopore in a MoS2 membrane, and the methyl-binding protein (MBP), MBD1x, to detect methylation on dsDNA. We show that the dsDNA translocation was effectively slowed down using an asymmetric concentration of buffer and explore the possibility of profiling the position of methylcytosines on the DNA strands as they translocate through the 2D membrane. Our findings advance us one step closer towards the possible use of nanopore sensing technology in medical applications such as cancer detection.

Modeling and Analysis of Intercalant Effects on Circular DNA Conformation.

The large-scale conformation of DNA molecules plays a critical role in many basic elements of cellular functionality and viability. By targeting the structural properties of DNA, many cancer drugs, such as anthracyclines, effectively inhibit tumor growth but can also produce dangerous side effects. To enhance the development of innovative medications, rapid screening of structural changes to DNA can provide important insight into their mechanism of interaction. In this study, we report changes to circular DNA conformation from intercalation with ethidium bromide using all-atom molecular dynamics simulations and characterized experimentally by translocation through a silicon nitride solid-state nanopore. Our measurements reveal three distinct current blockade levels and a 6-fold increase in translocation times for ethidium bromide-treated circular DNA as compared to untreated circular DNA. We attribute these increases to changes in the supercoiled configuration hypothesized to be branched or looped structures formed in the circular DNA molecule. Further evidence of the conformational changes is demonstrated by qualitative atomic force microscopy analysis. These results expand the current methodology for predicting and characterizing DNA tertiary structure and advance nanopore technology as a platform for deciphering structural changes of other important biomolecules.

Magnetophoretic-based microfluidic device for DNA Concentration.

Nucleic acids serve as biomarkers of disease and it is highly desirable to develop approaches to extract small number of such genomic extracts from human bodily fluids. Magnetic particles-based nucleic acid extraction is widely used for concentration of small amount of samples and is followed by DNA amplification in specific assays. However, approaches to integrate such magnetic particles based capture with micro and nanofluidic based assays are still lacking. In this report, we demonstrate a magnetophoretic-based approach for target-specific DNA extraction and concentration within a microfluidic device. This device features a large chamber for reducing flow velocity and an array of µ-magnets for enhancing magnetic flux density. With this strategy, the device is able to collect up to 95 % of the magnetic particles from the fluidic flow and to concentrate these magnetic particles in a collection region. Then an enzymatic reaction is used to detach the DNA from the magnetic particles within the microfluidic device, making the DNA available for subsequent analysis. Concentrations of over 1000-fold for 90 bp dsDNA molecules is demonstrated. This strategy can bridge the gap between detection of low concentration analytes from clinical samples and a range of micro and nanofluidic sensors and devices including nanopores, nano-cantilevers, and nanowires.

Slowing DNA Transport Using Graphene-DNA Interactions.

Slowing down DNA translocation speed in a nanopore is essential to ensuring reliable resolution of individual bases. Thin membrane materials enhance spatial resolution but simultaneously reduce the temporal resolution as the molecules translocate far too quickly. In this study, the effect of exposed graphene layers on the transport dynamics of both single (ssDNA) and double-stranded DNA (dsDNA) through nanopores is examined. Nanopore devices with various combinations of graphene and Al2O3 dielectric layers in stacked membrane structures are fabricated. Slow translocations of ssDNA in nanopores drilled in membranes with layers of graphene are reported. The increased hydrophobic interactions between the ssDNA and the graphene layers could explain this phenomenon. Further confirmation of the hydrophobic origins of these interactions is obtained through reporting significantly faster translocations of dsDNA through these graphene layered membranes. Molecular dynamics simulations confirm the preferential interactions of DNA with the graphene layers as compared to the dielectric layer verifying the experimental findings. Based on our findings, we propose that the integration of multiple stacked graphene layers could slow down DNA enough to enable the identification of nucleobases.

Nanopore-based assay for detection of methylation in double-stranded DNA fragments.

DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the CpG dyad in DNA fragments and could someday profile the position of methylated CpG sites on DNA fragments.

Electron beam induced local crystallization of HfO2 nanopores for biosensing applications.

We report the development of single, locally crystallized nanopores in HfO2 membranes for biosensing applications. HfO2 is chosen for its isoelectric point of 7.0, mechanical and chemical stability in solution, and for its potential as a high-k material for nanopore ionic field effect transistor applications. The HfO2 membrane is deposited on a graphene layer suspended over a 300 nm FIB hole, where graphene is used as the mechanical support. Exposure of the membrane to a focused electron beam causes crystallization in the vicinity of the nanopore during pore formation. We investigate the effects of crystallization on the electrical and surface properties of HfO2 films. Our surface analysis of HfO2 reveals improved hydrophilicity of crystallized HfO2, a notable advantage over the hydrophobicity of as-deposited HfO2. We also demonstrate detection of dsDNA translocation through HfO2 nanopores under various applied bias levels. In addition, our device architecture also presents a promising first step toward the realization of high-k HfO2 nanopore transistors.

Direct visualization of single-molecule translocations through synthetic nanopores comparable in size to a molecule.

A nanopore is the ultimate analytical tool. It can be used to detect DNA, RNA, oligonucleotides, and proteins with submolecular sensitivity. This extreme sensitivity is derived from the electric signal associated with the occlusion that develops during the translocation of the analyte across a membrane through a pore immersed in electrolyte. A larger occluded volume results in an improvement in the signal-to-noise ratio, and so the pore geometry should be made comparable to the size of the target molecule. However, the pore geometry also affects the electric field, the charge density, the electro-osmotic flow, the capture volume, and the response time. Seeking an optimal pore geometry, we tracked the molecular motion in three dimensions with high resolution, visualizing with confocal microscopy the fluorescence associated with DNA translocating through nanopores with diameters comparable to the double helix, while simultaneously measuring the pore current. Measurements reveal single molecules translocating across the membrane through the pore commensurate with the observation of a current blockade. To explain the motion of the molecule near the pore, finite-element simulations were employed that account for diffusion, electrophoresis, and the electro-osmotic flow. According to this analysis, detection using a nanopore comparable in diameter to the double helix represents a compromise between sensitivity, capture volume, the minimum detectable concentration, and response time.

Detection and quantification of methylation in DNA using solid-state nanopores.

Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores. Methylation is detected by selectively labeling methylation sites with MBD1 (MBD-1x) proteins, the complex inducing a 3 fold increase in ionic blockage current relative to unmethylated DNA. Furthermore, the discrimination of methylated and unmethylated DNA is demonstrated in the presence of only a single bound protein, thereby giving a resolution of a single methylated CpG dinucleotide. The extent of methylation of a target molecule could also be coarsely quantified using this novel approach. This nanopore-based methylation sensitive assay circumvents the need for bisulfite conversion, fluorescent labeling, and PCR and could therefore prove very useful in studying the role of epigenetics in human disease.

Electrochemistry at the edge of a single graphene layer in a nanopore.

We study the electrochemistry of single layer graphene edges using a nanopore-based structure consisting of stacked graphene and Al(2)O(3) dielectric layers. Nanopores, with diameters ranging from 5 to 20 nm, are formed by an electron beam sculpting process on the stacked layers. This leads to a unique edge structure which, along with the atomically thin nature of the embedded graphene electrode, demonstrates electrochemical current densities as high as 1.2 × 10(4) A/cm(2). The graphene edge embedded structure offers a unique capability to study the electrochemical exchange at an individual graphene edge, isolated from the basal plane electrochemical activity. We also report ionic current modulation in the nanopore by biasing the embedded graphene terminal with respect to the electrodes in the fluid. The high electrochemical specific current density for a graphene nanopore-based device can have many applications in sensitive chemical and biological sensing, and energy storage devices.

Using a nanopore for single molecule detection and single cell transfection.

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

Single-molecule investigation of G-quadruplex using a nanopore sensor.

This review article introduces the nanopore single-molecule method for the study of G-quadruplex nucleic acid structures. Single G-quadruplexes can be trapped into a 2 nm protein pore embedded in the lipid bilayer membrane. The trapped G-quadruplex specifically blocks the current through the nanopore, creating a signature event for quantitative analysis of G-quadruplex properties, from cation-determined folding and unfolding kinetics to the interactions with the protein ligand. The nanopore single-molecule method is simple, accurate, and requires no labels. It can be used to evaluate G-quadruplex mechanisms and it may have applications in G-quadruplex-based biosensors, nanomachines, and nanostructure assembly.