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.     

Fabrication of nanofluidic biochips with nanochannels for applications in DNA analysis

With the development of nanotechnology, great progress has been made in the fabrication of nanochannels. Nanofluidic biochips based on nanochannel structures allow biomolecule transport, bioseparation, and biodetection. The domain applications of nanofluidic biochips with nanochannels are DNA stretching and separation. In this Review, the general fabrication methods for nanochannel structures and their applications in DNA analysis are discussed. These representative fabrication approaches include conventional photolithography, interference lithography, electron-beam lithography, nanoimprint lithography and polymer nanochannels. Other nanofabrication methods used to fabricate unique nanochannels, including sub-10-nm nanochannels, single nanochannels, and vertical nanochannels, are also mentioned. These nanofabrication methods provide an effective way to form nanoscale channel structures for nanofluidics and biosensor devices for DNA separation, detection, and sensing. The broad applications of nanochannels and future perspectives are also discussed.     

A dual-beam optical microscope for observation and cleavage of single DNA molecules

An integrated epifocal and evanescent-wave optical microscope has been developed for real-time observation and bond cleavage studies of single DNA molecules. Large genomic DNA is stretched in a laminar flow stream and is immobilized on a polylysine-coated glass surface by strong electrostatic interactions. Unlike previous single-molecule dynamics and single-enzyme studies, this work takes advantage of the elastic nature of double-stranded DNA and measures DNA relaxation events that are triggered by two phosphodiester (P-O) bond breaks. The ability to follow chemical reactions on individual DNA molecules opens new possibilities for DNA mapping and for studying DNA-protein interactions.     

Lipid-based passivation in nanofluidics

Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein-DNA interactions with high spatial and temporal resolution.     

High‐Throughput Block Optical DNA Sequence Identification

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label‐free optical DNA sequencing technique will require nanoscale focusing of light, a high‐throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the “fingerprinting region” from ≈400–1400 cm^?1. Here, surface‐enhanced Raman spectroscopy is used to demonstrate label‐free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer‐scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k‐mers. The content of each nucleotide in a DNA block can be a unique and high‐throughput method for identifying sequences, genes, and other biomarkers as an alternative to single‐letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high‐throughput block optical sequencing method with lossy genomic data compression using k‐mer identification from multiplexed optical data acquisition.     

Hydrogen peroxide sensing with horseradish peroxidase-modified polymer single conical nanochannels

Inspired from the funtioning and responsiveness of biological ion channels, researchers attempt to develop biosensing systems based on polymer and solid-state nanochannels. The applicability of these nanochannels for detection/sensing of any foreign analyte in the surrounding environment depends critically on the surface characteristics of the inner walls. Attaching recognition sites to the channel walls leads to the preparation of sensors targeted at a specific molecule. There are many nanochannel platforms for the detection of DNA and proteins, but only a few are capable of detecting small molecules. Here, we describe a nanochannel platform for the detection of hydrogen peroxide, H(2)O(2), which is not only a toxic waste product in the cellular systems but also a key player in the redox signaling pathways. The sensor is based on single conical nanochannels fabricated in an ion tracked polymer membrane. The inner walls of the channel are decorated with horseradish peroxidase (HRP) enzyme using carbodiimide coupling chemistry. The success of the HRP immobilization on the channel surface is confirmed by measuring the pH-dependent current-voltage (I-V) curves of the system. The reported HRP-nanochannel system detects nanomolar concentrations of H(2)O(2) with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The immobilized HRP enzyme is thus capable of inducing redox reactions in a subfemtoliter volume of single nanochannels. We demonstrate that functioning of the designed biosensor is reversible and can be used multiple times to detect H(2)O(2) at various concentrations.     

Stretching DNA in polymer nanochannels fabricated by thermal imprint in PMMA

We present results regarding the fast and inexpensive fabrication of polymer biochips for investigating the statics and dynamics of DNA confined in nanochannels. The biochips have been fabricated by means of nanoimprint lithography (NIL) in low molecular weight polymethyl methacrylate (PMMA) using a 4?inch diameter two-level hybrid stamp. The fluidic structures were sealed using thermal polymer fusion bonding. The stamp has nanometer-?and micrometer-sized protrusions defined in a thermally grown SiO(2) layer and the sol-gel process derived duromeric hybrid polymer Ormocomp, respectively. The stamp is compatible with molecular vapor deposition (MVD), used for applying a durable chlorosilane based antistiction coating, and allows for imprint up to a temperature of 270?°C. The extension of YOYO-1 stained T4 GT7 bacteriophage DNA inside the PMMA nanochannels has been experimentally investigated using epi-fluorescence microscopy. The measured average extension length amounts to 20% of the full contour length with a standard deviation of 4%. These results are in good agreement with results obtained by stretching DNA in conventional fused silica nanochannels.     

Nucleic Acid Nanostructures for Chemical and Biological Sensing

The nanoscale features of DNA have made it a useful molecule for bottom‐up construction of nanomaterials, for example, two‐ and three‐dimensional lattices, nanomachines, and nanodevices. One of the emerging applications of such DNA‐based nanostructures is in chemical and biological sensing, where they have proven to be cost‐effective, sensitive and have shown promise as point‐of‐care diagnostic tools. DNA is an ideal molecule for sensing not only because of its specificity but also because it is robust and can function under a broad range of biologically relevant temperatures and conditions. DNA nanostructure‐based sensors provide biocompatibility and highly specific detection based on the molecular recognition properties of DNA. They can be used for the detection of single nucleotide polymorphism and to sense pH both in solution and in cells. They have also been used to detect clinically relevant tumor biomarkers. In this review, recent advances in DNA‐based biosensors for pH, nucleic acids, tumor biomarkers and cancer cell detection are introduced. Some challenges that lie ahead for such biosensors to effectively compete with established technologies are also discussed.     

Rapid prototyping of nanofluidic systems using size-reduced electrospun nanofibers for biomolecular analysis

Biomolecular transport in nanofluidic confinement offers various means to investigate the behavior of biomolecules in their native aqueous environments, and to develop tools for diverse single-molecule manipulations. Recently, a number of simple nanofluidic fabrication techniques has been demonstrated that utilize electrospun nanofibers as a backbone structure. These techniques are limited by the arbitrary dimension of the resulting nanochannels due to the random nature of electrospinning. Here, a new method for fabricating nanofluidic systems from size-reduced electrospun nanofibers is reported and demonstrated. As it is demonstrated, this method uses the scanned electrospinning technique for generation of oriented sacrificial nanofibers and exposes these nanofibers to harsh, but isotropic etching/heating environments to reduce their cross-sectional dimension. The creation of various nanofluidic systems as small as 20 nm is demonstrated, and practical examples of single biomolecular handling, such as DNA elongation in nanochannels and fluorescence correlation spectroscopic analysis of biomolecules passing through nanochannels, are provided.     

DNA-functionalized nanochannels for SNP detection

We have developed ultrahigh density array of functionalized nanochannels by using a block copolymer having end di-COOH group. This approach provides a facile route for direct functionalization of wall surface of the nanochannels and immobilization site for molecular recognition agents (MRAs). By using overhanging single-stranded DNA as MRAs, the DNA-functionalized nanochannels showed high resolution to detect a single-base mismatch as well as to discriminate single-mismatched sequence at various locations by hybridization preference with MRAs.     

Tunable non-equilibrium gating of flexible DNA nanochannels in response to transport flux

Biological nanochannels made from proteins play a central role in cellular signalling. The rapid emergence of DNA nanotechnology in recent years has opened up the possibility of making similar nanochannels from DNA. Building on previous work on switchable DNA nanocompartment, we have constructed complex DNA nanosystems to investigate the gating behaviour of these nanochannels. Here we show that DNA nanochannels can be gated by stress exerted by permeating solute particles at non-equilibrium states due to the high flexibility of the nanochannels. This novel gating mechanism results in tunable ratchet-like transport of solute particles through the nanochannels. A simple model that couples non-equilibrium channel gating with transport flux can quantitatively explain a number of the phenomena we observe. With only one set of model parameters, we can reproduce diverse gating behaviours, modulated by an inherent gating threshold. This work could lead to the development of new devices based on DNA nanochannels.     

Combed single DNA molecules imaged by secondary ion mass spectrometry

Studies of replication, recombination, and rearrangements at the level of individual molecules of DNA are often limited by problems of resolution or of perturbations caused by the modifications that are needed for imaging. The Combing-Imaging by Secondary Ion Mass Spectrometry (SIMS) (CIS) method helps solve these problems by combining DNA combing, cesium flooding, and quantitative imaging via the NanoSIMS 50. We show here that CIS can reveal, on the 50 nm scale, individual DNA fibers labeled with different, nonradioactive isotopes and, moreover, that it can quantify these isotopes so as to detect and measure the length of one or more short nucleic acid fragments associated with a longer fiber.     

Nanostructures confined self-assembled in biomimetic nanochannels for enhancing the sensitivity of biological molecules response

The combination of biomimetic nanochannels and nanostructures provided a new idea for bioanalytical purposes. In this paper, we reported a self-assembled system based on the integration of anodic aluminum oxide (AAO) membranes and CuO method to fabricate CuO nanostructures in the confined area of AAO membranes. Furthermore, we investigated morphologies of the self-assembly systems by field emission scanning electron microscopy (FESEM) and characterized the type and content of microelements by energy dispersive spectrometer. Transmembrane ion current through nanochannels was tested by an electrochemical workstation, and optical properties of the self-assembly systems were characterized by ultraviolet–visible spectrophotometer. We could conclude from the results that the morphology and distribution status of CuO nanostructures are controllable, while ionic current through nanochannels and optical properties of self-assembled systems could be regulated by changing experimental parameters such as electrodeposition time, electrodeposition voltage or annealing temperature. On this base, we utilized the synergistic effect of nanochannels and nanostructures to enhance the sensitivity of biological molecules response. This study not only prepared CuO nanostructures in biomimetic nanochannels controllably, but also provided a novel application paradigm for nanochannels-nanostructures self-assembled system, which could be used for biological analysis and detection.     

Description of DNA molecular motion for nanotechnology applications

One of the most important quests in modern science is the ability to mimic DNA replication, this process provides opportunities to vastly improve current technology from data storage to fighting disease. How molecular processes govern DNA signalling is a key question to be answered for this endeavour to be achieved. One of the greatest difficulties with DNA manipulation has been to physically manipulate individual DNA molecules. In vivo there are a large variety of proteins present which are finely controlled by other proteins and signalling molecules to ensure DNA will coil when required and uncoil for replication when stimulated. Understanding the dynamics of motion of particles and molecules that participate in DNA processes is key to understanding and potentially mimicking these processes. In this review, we introduce the current knowledge with respect to molecular dynamics of motion and draw reference to DNA natural and artificial processes. We also discuss the motion mechanisms and how they might explain the efficiency of DNA signalling and replication processes.     

Labeling TiO2 Nanoparticles with Dyes for Optical Fluorescence Microscopy and Determination of TiO2–DNA Nanoconjugate Stability

Visualization of nanoparticles without intrinsic optical fluorescence properties is a significant problem when performing intracellular studies. Such is the case with titanium dioxide (TiO₂) nanoparticles. These nanoparticles, when electronically linked to single‐stranded DNA oligonucleotides, have been proposed to be used both as gene knockout devices and as possible tumor imaging agents. By interacting with complementary target sequences in living cells, these photoinducible TiO₂–DNA nanoconjugates have the potential to cleave intracellular genomic DNA in a sequence specific and inducible manner. The nanoconjugates also become detectable by magnetic resonance imaging with the addition of gadolinium Gd(III) contrast agents. Herein two approaches for labeling TiO₂ nanoparticles and TiO₂–DNA nanoconjugates with optically fluorescent agents are described. This permits direct quantification of fluorescently labeled TiO₂ nanoparticle uptake in a large population of living cells (>10⁴ cells). X‐ray fluorescence microscopy (XFM) is combined with fluorescent microscopy to determine the relative intracellular stability of the nanoconjugates and used to quantify intracellular nanoparticles. Imaging the DNA component of the TiO₂–DNA nanoconjugate by fluorescent confocal microscopy within the same cell shows an overlap with the titanium signal as mapped by XFM. This strongly implies the intracellular integrity of the TiO₂–DNA nanoconjugates in malignant cells.     

Flossing DNA in a Dual Nanopore Device

Solid‐state nanopores are a single‐molecule technique that can provide access to biomolecular information that is otherwise masked by ensemble averaging. A promising application uses pores and barcoding chemistries to map molecular motifs along single DNA molecules. Despite recent research breakthroughs, however, it remains challenging to overcome molecular noise to fully exploit single‐molecule data. Here, an active control technique termed “flossing” that uses a dual nanopore device is presented to trap a proteintagged DNA molecule and up to 100's of back‐and‐forth electrical scans of the molecule are performed in a few seconds. The protein motifs bound to 48.5 kb λ‐DNA are used as detectable features for active triggering of the bidirectional control. Molecular noise is suppressed by averaging the multiscan data to produce averaged intertag distance estimates that are comparable to their known values. Since nanopore feature‐mapping applications require DNA linearization when passing through the pore, a key advantage of flossing is that trans‐pore linearization is increased to >98% by the second scan, compared to 35% for single nanopore passage of the same set of molecules. In concert with barcoding methods, the dual‐pore flossing technique could enable genome mapping and structural variation applications, or mapping loci of epigenetic relevance.     

Identifying DNA methylation in a nanochannel

DNA methylation is a stable epigenetic modification, which is well known to be involved in gene expression regulation. In general, however, analyzing DNA methylation requires rather time consuming processes (24–96 h) via DNA replication and protein modification. Here we demonstrate a methodology to analyze DNA methylation at a single DNA molecule level without any protein modifications by measuring the contracted length and relaxation time of DNA within a nanochannel. Our methodology is based on the fact that methylation makes DNA molecules stiffer, resulting in a longer contracted length and a longer relaxation time (a slower contraction rate). The present methodology offers a promising way to identify DNA methylation without any protein modification at a single DNA molecule level within 2 h.     

Electrochemically replicated smooth aluminum foils for anodic alumina nanochannel arrays

A fast electrochemical replication technique has been developed to fabricate large-scale ultra-smooth aluminum foils by exploiting readily available large-scale smooth silicon wafers as the masters. Since the adhesion of aluminum on silicon depends on the time of surface pretreatment in water, it is possible to either detach the replicated aluminum from the silicon master without damaging the replicated aluminum and master or integrate the aluminum film to the silicon substrate. Replicated ultra-smooth aluminum foils are used for the growth of both self-organized and lithographically guided long-range ordered arrays of anodic alumina nanochannels without any polishing pretreatment.     

Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles

Nanometer-sized fluorescent particles (latex nanobeads) have been covalently linked to DNA binding proteins to probe specific sequences on stretched single DNA molecules. In comparison with single organic fluorophores, these nanoparticle probes are brighter, are more stable against photobleaching, and do not suffer from intermittent on/off light emission (blinking). Specifically, we demonstrate that the site-specific restriction enzyme EcoRI can be conjugated to 20-nm fluorescent nanoparticles and that the resulting nanoconjugates display DNA binding and cleavage activities of the native enzyme. In the absence of cofactor magnesium ions, the EcoRI conjugates bind to specific sequences on double-stranded DNA but do not initiate enzymatic cutting. For single DNA molecules that are stretched and immobilized on a solid surface, nanoparticles bound at specific sites can be directly visualized by multicolor fluorescence microscopy. Direct observation of site-specific probes on single DNA molecules opens new possibilities in optical gene mapping and in the fundamental study of DNA-protein interactions.     

DNA‐SMART: Biopatterned Polymer Film Microchannels for Selective Immobilization of Proteins and Cells

A novel SMART module, dubbed “DNA‐SMART” (DNA substrate modification and replication by thermoforming) is reported, where polymer films are premodified with single‐stranded DNA capture strands, microthermoformed into 3D structures, and postmodified with complementary DNA‐protein conjugates to realize complex biologically active surfaces within microfluidic devices. As a proof of feasibility, it is demonstrated that microchannels presenting three different proteins on their inner curvilinear surface can be used for selective capture of cells under flow conditions.