Nanochannel Arrays Research Articles

Embedding 1D Conducting Channels into 3D Isoporous Polymer Films for High‐Performance Humidity Sensing

AbstractIsoporous block copolymer (BCP) films have received exponential interest as highly selective membranes, stemming from their unique morphological features, but their applications in functional devices remain to be realized. Now single‐walled carbon nanotubes (CNTs) were efficiently incorporated into isoporous block copolymer films for chemiresistive sensing at room temperature. Leveraging the efficient charge extraction ability of CNTs together with nanochannel arrays aligned perpendicular to the surface of the films, an ultrafast response time of 0.3 s was achieved for humidity detection with a sensor response of about 800 on changing humidity from 10 % to 95 %. Furthermore, the sensor also responds to various organic vapors, underscoring its promising detection capability.

Embedding 1D Conducting Channels into 3D Isoporous Polymer Films for High-Performance Humidity Sensing.

Isoporous block copolymer (BCP) films have received exponential interest as highly selective membranes, stemming from their unique morphological features, but their applications in functional devices remain to be realized. Now single-walled carbon nanotubes (CNTs) were efficiently incorporated into isoporous block copolymer films for chemiresistive sensing at room temperature. Leveraging the efficient charge extraction ability of CNTs together with nanochannel arrays aligned perpendicular to the surface of the films, an ultrafast response time of 0.3 s was achieved for humidity detection with a sensor response of about 800 on changing humidity from 10 % to 95 %. Furthermore, the sensor also responds to various organic vapors, underscoring its promising detection capability.

Abstract 414: Broad range chromosomal abnormality detection through Bionano genome mapping

Abstract Current methods for detection of balanced structural variation can be broken down into two categories: traditional cytogenetics and molecular methods. Cytogenetics may include chromosomal karyotyping, fluorescence in situ hybridization (FISH), chromosomal microarray and adaption of them. Molecular methods primarily include NGS sequencing based methods. Bionano genome mapping, an optical mapping approach in a NanoChannel array system, is a method that combines the advantages of different categories while solving many of the limitations. Compared to cytogenetics, Bionano mapping is high throughput and removes manual interpretation, it also has much higher resolution, detecting balanced events as small as about 30 kbp compared to multi-megabases needed for cytogenetic approaches, and unbalanced events starting at 1 kbp. NGS based methods often are limited by read lengths that cannot provide unambiguous information across repeat elements longer than individual reads. This limitation results in significantly reduced sensitivity for balanced variation and abnormalities as well as for insertions and even some categories of larger deletions. This is particularly true in highly medically relevant locations where segmental duplications mediate chromosomal abnormalities. Genome mapping using Bionano Genomics' Saphyr System offers high-throughput, genome-wide visualization of extremely long DNA molecules in their native form. It allows researchers to interrogate genomic structural variations (SVs) in the range of one kilobase pair and above. We present several in silico and biological validation experiments that demonstrate the sensitivity and specificity of Bionano mapping for the detection of insertions, deletions, inversions and translocations compared to benchmark studies using short read and long read sequencing. We also show a new direct labeling method that dramatically improves assembly contiguity such that chromosome arms can be assembled into single maps. Bionano mapping is a fast and cost effective method for the detection of a broad range of traditionally refractory SVs across the human genome. Citation Format: Alex R. Hastie. Broad range chromosomal abnormality detection through Bionano genome mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 414.

Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays.

The epigenetic mark 5-hydroxymethylcytosine (5-hmC) is a distinct product of active DNA demethylation that is linked to gene regulation, development, and disease. In particular, 5-hmC levels dramatically decline in many cancers, potentially serving as an epigenetic biomarker. The noise associated with next-generation 5-hmC sequencing hinders reliable analysis of low 5-hmC containing tissues such as blood and malignant tumors. Additionally, genome-wide 5-hmC profiles generated by short-read sequencing are limited in providing long-range epigenetic information relevant to highly variable genomic regions, such as the 3.7 Mbp disease-related Human Leukocyte Antigen (HLA) region. We present a long-read, highly sensitive single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5-hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long single-molecule read-length of 100 kbp to 1 Mbp produces 5-hmC profiles across variable genomic regions that failed to show up in the sequencing data. In addition, optical 5-hmC mapping shows a strong correlation between the 5-hmC density in gene bodies and the corresponding level of gene expression. The single-molecule concept provides information on the distribution and coexistence of 5-hmC signals at multiple genomic loci on the same genomic DNA molecule, revealing long-range correlations and cell-to-cell epigenetic variation.

Solid-Liquid Electrolyte as a Nanoion Modulator for Dendrite-Free Lithium Anodes.

Rechargeable lithium (Li) metal batteries are considered the most promising of Li-based energy storage technologies. However, tree-like dendrite produced by irregular Li+ electrodeposition restricts it wide applications. Herein, based on a cation-microphase-regulation strategy, we create solid-liquid electrolytes (SLEs) by absorbing commercial liquid electrolytes into polyethylene glycol (PEG) engineered nanoporous Al2O3 ceramic membranes. By means of molecular dynamics simulations and comprehensive experiments, we show that Li ions are regulated and promoted in the two microphases, the channel phase and nonchannel phase, respectively. The channel phase can achieve homogeneous Li+ flux distribution by multiple mechanisms, including its uniform array of nanochannels and ability to suppress lateral dendrite growth by its high modulus. In the nonchannel phase, PEG chains swollen by electrolyte facilitate desolvation and fast conduction of Li+. As a result, the studied SLEs exhibit high ionic conductivity, low interfacial resistance, and the unique ability to stabilize deposition at the Li anode. By means of galvanostatic cycling studies in symmetric Li cells and Li/Li4Ti5O12 cells, we further show that the materials open a path to Li metal batteries with excellent cycling performance.

Thermo and pH Dual – Actuating Smart Porous Anodic Aluminum for Controllable Drug Release

AbstractA highly effective dual‐stimuli responsive porous anodic aluminum (PAA) nanomembrane is designed and fabricated for controllable and sustainable drug release. By a two‐step electrochemical anodization process, the nanochannel arrays in PAA membrane are divided into two separated parts. The first part is modified by copolymer‐liposome (colipid), which acts as the thermo and pH dual‐stimuli responsive capping material. The other part is used as the drug loading core. The liposome in capping layer has excellent biocompatibility, and the nanochannels array can provide a high drug loading capacity. Taking dexamethasone as the model drug, the experiment results show that the fabricated nanochannels can be well tuned to open or close state upon pH or temperature stimuli, which allows the controllable and sustainable drug release. The present work is of significant importance for constructing smart and intelligent drug carrier and release systems for clinical applications.

Ionic Transport in Aptamer Functionalized Nanochannel Array

We report theoretical and experimental results of the influence of surface immobilized aptamers on ionic transport through nanochannels. The ionic transport in a polyelectrolyte functionalized nanochannel may be modified either by electrostatic mechanisms that involve changes in double layer formed near the pore walls or through steric hindrance of the surface chains on the transport of solution ions. Arrays of well-defined nanochannels are fabricated using two-step anodization process of aluminum on ITO coated glass substrate. The anodization voltage and electrolyte are chosen to achieve the desired nanochannel radius and distribution. The alumina nanochannels are functionalized with thrombin binding DNA aptamers (TBA) to enable the formation of monomolecular films of polyelectrolytes on the nanochannel surface. The functionalized nanochannel arrays are then exposed to different concentrations of α-thrombin and change in impedance across the nanochannel array are characterized in order to identify the influence of protein/aptamer binding on the ionic conduction in the nanochannels. Both DNA aptamers (specific to thrombin) and α-thrombin are charged species and their interactions produce modulation of surface charges and modify the net ionic conductance across the nanochannel. We also characterized changes in ionic conductance in presence of γ-thrombin, an uncharged protein that has similar physiological structure as α-thrombin but no affinity towards TBA. Ionic and solution transport in the nanochannel is modeled using coupled Poisson-Nernst-Planck equation and Navier-Stokes equations. The conformation and charge density of aptamer monomolecular films on the nanochannel surface was modeled based on molecular dynamics simulation results of aptamer and thrombin/aptamer complexes (Gosai et al., 2016). The predicted changes in ionic conductance are compared to experimental results to identify the influence of electrostatic and steric interactions on the ion transport in polyelectrolyte functionalized nanochannel. The results show that aptamer functionalized alumina nanochannel have potential to be used for the development of disposable and point of care devices by employing relatively facile and inexpensive fabrication methodologies. Reference: Gosai, A., Ma, X., Balasubramanian, G., Shrotriya, P., 2016. Electrical Stimulus Controlled Binding/Unbinding of Human Thrombin-Aptamer Complex. Sci. Rep. 6, 37449. doi:10.1038/srep37449

Tunable Confinement for Bridging Single-Cell Manipulation and Single-Molecule DNA Linearization.

DNA linearization by nanoconfinement has offered a new avenue toward large-scale genome mapping. The ability to smoothly interface the widely different length scales from cell manipulation to DNA linearization is critical to the development of single-cell genomic mapping or sequencing technologies. Conventional nanochannel technologies for DNA analysis suffer from complex fabrication procedures, DNA stacking at the nanochannel entrance, and inefficient solution exchange. In this work, a dynamic and tunable confinement strategy is developed to manipulate and linearize genomic-length DNA molecules from a single cell. By leveraging pneumatic microvalve control and elastomeric collapse, an array of nanochannels with confining dimension down to 20 nm and length up to sub-millimeter is created and can be dynamically tuned in size. The curved edges of the microvalve form gradual transitions from microscale to nanoscale confinement, smoothly facilitating DNA entry into the nanochannels. A unified micro/nanofluidic device that integrates single-cell trapping and lysis, DNA extraction, purification, labeling, and linearization is developed based on dynamically controllable nanochannels. Mbp-long DNA molecules are extracted directly from a single cell and in situ linearized in the nanochannels. The device provides a facile and promising platform to achieve the ultimate goal of single-cell, single-genome analysis.

Characterization and electrochemical response of DNA functionalized 2 nm gold nanoparticles confined in a nanochannel array

Polyvalent gold nanoparticle oligonucleotide conjugates are subject of intense research. Even though 2nm diameter AuNPs have been previously modified with DNA, little is known about their structure and electrochemical behavior. In this work, we examine the influence of different surface modification strategies on the interplay between the meso-organization and the molecular recognition properties of a 27-mer DNA strand. This DNA strand is functionalized with different sulfur-containing moieties and immobilized on 2nm gold nanoparticles confined on a nanoporous alumina, working the whole system as an electrode array. Surface coverages were determined by EXAFS and the performance as recognition elements for impedance-based sensors is evaluated. Our results prove that low DNA coverages on the confined nanoparticles prompt to a more sensitive response, showing the relevance in avoiding the DNA strand overcrowding. The system was able to determine a concentration as low as 100pM of the complementary strand, thus introducing the foundations for the construction of label-free genosensors at the nanometer scale.

Non-lithographic nanofluidic channels with precisely controlled circular cross sections.

Nanofluidic channels have received growing interest due to their potential for applications in the manipulation of nanometric objects, such as DNA, proteins, viruses, exosomes, and nanoparticles. Although significant advances in nanolithography-based fabrication techniques over the past few decades have allowed us to explore novel nanofluidic transport phenomena and unique applications, the development of new technologies enabling the low-cost preparation of nanochannels with controllable and reproducible shapes and dimensions is still lacking. Thus, we herein report the application of a nanofiber printed using a near-field electrospinning method as a sacrificial mold for the preparation of polydimethylsiloxane nanochannels with circular cross sections. Control of the size and shape of these nanochannels allowed the preparation of nanochannels with channel widths ranging from 70–368 nm and height-to-width ratios of 0.19–1.00. Capillary filling tests confirmed the excellent uniformity and reproducibility of the nanochannels. These results therefore are expected to inspire novel nanofluidic studies due to the simple and low-cost nature of this fabrication process, which allows precise control of the shape and dimensions of the circular cross section.

Single-crystalline integrated 4H-SiC nanochannel array electrode: toward high-performance capacitive energy storage for robust wide-temperature operation

The electrode nearly full-featured for robust wide-temperature operation based on single-crystalline integrated 4H-SiC nanochannel arrays was explored.

Asymmetric Nanochannel-Ionchannel Hybrid for Ultrasensitive and Label-Free Detection of Copper Ions in Blood.

Nanochannel/nanopre based analysis methods have attracted increasing interest in recent years due to their exquisite ability to reveal changes in molecular volume. In this work, a highly asymmetric nanochannel-ionchannel hybrid coupled with an electrochemical technique was developed for copper ion (Cu2+) detection. Polyglutamic acid (PGA) was modified in a nanochannel array of porous anodic alumina (PAA). When different concentrations of Cu2+ were introduced into the nanochannel-ionchannel hybrid in a neutral environment, a Cu2+-PGA chelation reaction occurs, resulting in varied current-potential (I-V) properties of the nanochannel-ionchannel hybrid. When PAA was immersed in a low pH solution, the Cu2+-PGA complex dissociated. On the basis of the change in ionic current, a label-free assay for Cu2+ was achieved along with the ability to regenerate and reuse the constructed platform. Because of the unique mass transfer property of the nanochannel-ionchannel hybrid combined with the highly amplified ionic current magnitude of the nanochannel array, significantly increased assay sensitivity was achieved, as expected. To evaluate the applicability of the present methodology for detecting Cu2+ in a real sample, the Cu2+ content in real blood samples was analyzed. The results demonstrate that the present method shows excellent selectivity with high sensitivity toward Cu2+ detection in real blood samples.

Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis

BackgroundMassively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient’s phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100–300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations.MethodsTo overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene.ResultsWe identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45–250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing.ConclusionsWe showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.

SP1 based self-assembled selective molecular nanochannels

Controlling the permeability and porosity of an inorganic layer using biomolecule building blocks has raised interest for nanotechnological applications. The challenge lies mostly in the fabrication, usually a long, expensive and tedious process, involving many steps. Using biomaterials for this purpose is highly appealing; due to both ease of fabrication and the final output, that contains a bioelement. The biomolecule, specifically, stable protein 1 (SP1), serving as the scaffold for our pattern, is of great stability and durability, and presents size, charge and structural selectivity towards electroactive species. Here, we demonstrate the ability of SP1 to form a rigid template within a sol-gel matrix, allowing selective electron transfer to the gold electrode. Specifically, a thiolated SP1 was first adsorbed on a gold surface followed by filling the non-occupied areas by sol-gel. The latter was electrochemically deposited. The various steps were carefully characterized. Finally, we studied the electrochemistry of numerous redox couple at the Au/SP1/sol-gel interface and found that the nanochannel array shows charge and structural selectivity, which is based on the interactions between the redox species and the functionalities of SP1. The resulted surface shows promise towards electrochemical sensing applications.

High-throughput single-molecule telomere characterization.

We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30–35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines.

A multiscale fluidic device for the study of dendrite-mediated cell to cell communication.

Many cell types communicate by means of dendritic extensions via a multi-tiered set of geometric and chemical cues. Until recently, mimicking the compartmentalized in vivo cellular environment of dendrite-expressing cells such as osteocytes and motor neurons in a spatially and temporally controllable manner was limited by the challenges of in vitro device fabrication at submicron scales. Utilizing the improved resolution of current fabrication technology, we have designed a multiscale device, the Macro-micro-nano system, or Mμn, composed of two distinct cell-seeding and interrogation compartments separated by a nanochannel array. The array enables dendrite ingrowth, while providing a mechanism for fluidic sequestration and/or temporally-mediated diffusible signaling between cell populations. Modeling of the Mμn system predicted the ability to isolate diffusible signals, namely ATP. Empirical diffusion studies verified computational modeling. In addition, cell viability, dendrite interaction with the nanoarray, and cellular purinergic response to heat shock were experimentally evaluated within the device for both osteocytes and motor neurons. Our results describe a novel in vitro system in which dendrite-expressing cell types can be studied within nano-environments that mimic in vivo conditions. In particular, the Mμn system enables real-time observation of cell to cell communication between cell populations in distinct, but fluidically coupled regions.

Nanochannel Arrays for Molecular Sieving and Electrochemical Analysis by Nanosphere Lithography Templated Graphoepitaxy of Block Copolymers.

The ability to design, fabricate, and manipulate materials at the nanoscale is fundamental to the quest to develop technologies to assemble nanometer-scale pieces into larger-scale components and materials, thereby transferring unique nanometer-scale properties to macroscopic objects. In this work, we describe a new approach to the fabrication of highly ordered, ultrahigh density nanochannel arrays that employs nanosphere lithography to template the graphoepitaxy of polystyrene-polydimethylsiloxane, diblock copolymers. By optimizing the well-controlled solvent vapor annealing, overcoating conditions, and the subsequent reactive ion etching processes, silica nanochannel (SNC) arrays with areal densities, ρA, approaching 1000 elements μm-2, are obtained over macroscopic scales. The integrity and functionality of the SNC arrays was tested by using them as permselective ion barriers to nanopore-confined disk electrodes. The nanochannels allow cations to pass to the disk electrode but reject anions, as demonstrated by cyclic voltammetry. This ion gating behavior can be reversed from cation-permselective to anion-permselective by chemically inverting the surface charge from negative to positive. Furthermore, the conformal SNC array structures obtained could easily be lifted, detached, and transferred to another substrate, preserving the hierarchical organization while transferring the nanostructure-derived properties to a different substrate. These results demonstrate how nanoscale behavior can be replicated over macroscale distances, using electrochemical analysis as a model.

Assessing Antibiotic Permeability of Gram-Negative Bacteria via Nanofluidics

While antibiotic resistance is increasing rapidly, drug discovery has proven to be extremely difficult. Antibiotic resistance transforms some bacterial infections into deadly medical conditions. A significant challenge in antibiotic discovery is designing potent molecules that enter Gram-negative bacteria and also avoid active efflux mechanisms. Critical analysis in rational drug design has been hindered by the lack of effective analytical tools to analyze the bacterial membrane permeability of small molecules. We design, fabricate, and characterize the nanofluidic device that actively loads more than 200 single bacterial cells in a nanochannel array. We demonstrate a gigaohm seal between the nanochannel walls and the loaded bacteria, restricting small molecule transport to only occur through the bacterial cell envelope. Quantitation of clindamycin translocation through wild-type and efflux-deficient (ΔtolC) Escherichia coli strains via nanofluidic-interfaced liquid chromatography mass spectrometry shows higher levels of translocation for wild-type E. coli than for an efflux-deficient strain. We believe that the assessment of compound permeability in Gram-negative bacteria via the nanofluidic analysis platform will be an impactful tool for compound permeation and efflux studies in bacteria to assist rational antibiotic design.

Label-free monitoring of the thrombin–aptamer recognition reaction using an array of nanochannels coupled with electrochemical detection

A sensitive and label-free method of monitoring the thrombin–aptamer recognition reaction has been developed using an array of nanochannels coupled with an electrochemical detection technique. Due to the highly amplified ion current produced by an array of nanochannels compared to a single nanochannel/pore, a significant increase in detection sensitivity has been achieved.

An advanced reference genome of Trifolium subterraneum L. reveals genes related to agronomic performance.

SummarySubterranean clover is an important annual forage legume, whose diploidy and inbreeding nature make it an ideal model for genomic analysis in Trifolium. We reported a draft genome assembly of the subterranean clover TSUd_r1.1. Here we evaluate genome mapping on nanochannel arrays and generation of a transcriptome atlas across tissues to advance the assembly and gene annotation. Using a BioNano‐based assembly spanning 512 Mb (93% genome coverage), we validated the draft assembly, anchored unplaced contigs and resolved misassemblies. Multiple contigs (264) from the draft assembly coalesced into 97 super‐scaffolds (43% of genome). Sequences longer than >1 Mb increased from 40 to 189 Mb giving 1.4‐fold increase in N50 with total genome in pseudomolecules improved from 73 to 80%. The advanced assembly was re‐annotated using transcriptome atlas data to contain 31 272 protein‐coding genes capturing >96% of the gene content. Functional characterization and GO enrichment confirmed gene expression for response to water deprivation, flavonoid biosynthesis and embryo development ending in seed dormancy, reflecting adaptation to the harsh Mediterranean environment. Comparative analyses across Papilionoideae identified 24 893 Trifolium‐specific and 6325 subterranean‐clover‐specific genes that could be mined further for traits such as geocarpy and grazing tolerance. Eight key traits, including persistence, improved livestock health by isoflavonoid production in addition to important agro‐morphological traits, were fine‐mapped on the high‐density SNP linkage map anchored to the assembly. This new genomic information is crucial to identify loci governing traits allowing marker‐assisted breeding, comparative mapping and identification of tissue‐specific gene promoters for biotechnological improvement of forage legumes.