Plasmonic‐Nanopore Biosensors for Superior Single‐Molecule Detection

Plasmonic and nanopore sensors have separately received much attention for achieving single‐molecule precision. A plasmonic “hotspot” confines and enhances optical excitation at the nanometer length scale sufficient to optically detect surface–analyte interactions. A nanopore biosensor actively funnels and threads analytes through a molecular‐scale aperture, wherein they are interrogated by electrical or optical means. Recently, solid‐state plasmonic and nanopore structures have been integrated within monolithic devices that address fundamental challenges in each of the individual sensing methods and offer complimentary improvements in overall single‐molecule sensitivity, detection rates, dwell time and scalability. Here, the physical phenomena and sensing principles of plasmonic and nanopore sensing are summarized to highlight the novel complementarity in dovetailing these techniques for vastly improved single‐molecule sensing. A literature review of recent plasmonic nanopore devices is then presented to delineate methods for solid‐state fabrication of a range of hybrid device formats, evaluate the progress and challenges in the detection of unlabeled and labeled analyte, and assess the impact and utility of localized plasmonic heating. Finally, future directions and applications inspired by the present state of the art are discussed.     

Perspectives and Challenges of Emerging Single‐Molecule DNA Sequencing Technologies

The growing demand for analysis of the genomes of many species and cancers, for understanding the role of genetic variation among individuals in disease, and with the ultimate goal of deciphering individual human genomes has led to the development of non‐Sanger reaction‐based technologies towards rapid and inexpensive DNA sequencing. Recent advancements in new DNA sequencing technologies are changing the scientific horizon by dramatically accelerating biological and biomedical research and promising an era of personalized medicine for improved human health. Two single‐molecule sequencing technologies based on fluorescence detection are already in a working state. The newly launched and emerging single‐molecule DNA sequencing approaches are reviewed here. The current challenges of these technologies and potential methods of overcoming these challenges are critically discussed. Further research and development of single‐molecule sequencing will allow researchers to gather nearly error‐free genomic data in a timely and inexpensive manner.

     

Fabrication of Metallized Nanopores in Silicon Nitride Membranes for Single‐Molecule Sensing

The fabrication and characterization of a metallized nanopore structure for the sensing of single molecules is described. Pores of varying diameters (>10 nm) are patterned into free‐standing silicon nitride membranes by electron‐beam lithography and reactive ion etching. Structural characterization by transmission electron microscopy (TEM) and tomography reveals a conical pore shape with a 40° aperture. Metal films of Ti/Au are vapor deposited and the pore shape and shrinking are studied as a function of evaporated film thickness. TEM tomography analysis confirms metalization of the inner pore walls as well as conservation of the conical pore shape. In electrical measurements of the transpore current in aqueous electrolyte solution, the pores feature very low noise. The applicability of the metallized pores for stochastic sensing is demonstrated in real‐time translocation experiments of single λ‐DNA molecules. We observe exceptionally long‐lasting current blockades with a fine structure of distinct current levels, suggesting an attractive interaction between the DNA and the PEGylated metallic pore walls.     

Cross‐Talk Between Ionic and Nanoribbon Current Signals in Graphene Nanoribbon‐Nanopore Sensors for Single‐Molecule Detection

Nanopores are now being used not only as an ionic current sensor but also as a means to localize molecules near alternative sensors with higher sensitivity and/or selectivity. One example is a solid‐state nanopore embedded in a graphene nanoribbon (GNR) transistor. Such a device possesses the high conductivity needed for higher bandwidth measurements and, because of its single‐atomic‐layer thickness, can improve the spatial resolution of the measurement. Here measurements of ionic current through the nanopore are shown during double‐stranded DNA (dsDNA) translocation, along with the simultaneous response of the neighboring GNR due to changes in the surrounding electric potential. Cross‐talk originating from capacitive coupling between the two measurement channels is observed, resulting in a transient response in the GNR during DNA translocation; however, a modulation in device conductivity is not observed via an electric‐field‐effect response during DNA translocation. A field‐effect response would scale with GNR source–drain voltage (V_ds), whereas the capacitive coupling does not scale with V_ds. In order to take advantage of the high bandwidth potential of such sensors, the field‐effect response must be enhanced. Potential field calculations are presented to outline a phase diagram for detection within the device parameter space, charting a roadmap for future optimization of such devices.     

Surface Modification of Solid‐State Nanopores for Sticky‐Free Translocation of Single‐Stranded DNA

Nanopore technology is one of the most promising approaches for fast and low‐cost DNA sequencing application. Single‐stranded DNA detection is primary objective in such device, while solid‐state nanopores remain less explored than their biological counterparts due to bio‐molecule clogging issue caused by surface interaction between DNA and nanopore wall. By surface coating a layer of polyethylene glycol (PEG), solid‐state nanopore can achieve long lifetime for single‐stranded DNA sticky‐free translocation at pH 11.5. Associated with elimination of non‐specific binding of molecule, PEG coated nanopore presents new surface characteristic as less hydrophility, lower 1/f noise, and passivated surface charge responsiveness on pH. Meanwhile, conductance blockage of single‐stranded DNA is found to be deeper than double‐stranded DNA, which can be well described by a string of blobs model for a quasi‐equilibrium state polymer in constraint space.     

Micrometer‐sized DNA–Single‐Fluorophore–DNA Supramolecule: Synthesis and Single‐Molecule Characterization

The synthesis of single‐fluorophore‐bis(micrometer‐sized DNA) triblock supramolecules and the optical and structural characterization of the construct at the single‐molecule level is reported. A fluorophore‐bis(oligodeoxynucleotide) triblock is synthesized via the amide‐coupling reaction. Subsequent protocols of DNA hybridization/ligation are developed to form the supramolecular triblock structure with λ‐DNA fragments on the micrometer length scale. The successful synthesis of the micrometer‐sized DNA–single‐fluorophore–DNA supramolecule is confirmed by agarose gel electrophoresis with fluorescence imaging under UV excitation. Single triblock structures are directly imaged by combined scanning force microscopy and single‐molecule fluorescence microscopy, and provide unambiguous confirmation of the existence of the single fluorophore inserted in the middle of the long DNA. This type of triblock structure is a step closer to providing a scaffold for single‐molecule electronic devices after metallization of the DNAs.     

Nanopore Biosensors: Monitoring of an ATP‐Binding Aptamer and its Conformational Changes Using an α‐Hemolysin Nanopore (Small 1/2011)

An aptamer is a specific oligonucleotide sequence that spontaneously forms a secondary structure capable of selectively binding an analyte. An aptamer’s conformation is the key to specific binding of a target molecule, even in the case of very closely related targets. Nanopores are a sensitive tool for the single‐molecule analysis of DNA, peptides, and proteins transporting through the pore. Herein, a single α‐hemolysin natural nanopore is utilized to sense the conformational changes of an adenosine 5’‐triphosphate (ATP)‐binding aptamer (ABA). The known DNA sequence of the ABA is used as a model to develop real‐time monitoring of molecular conformational changes that occur by binding targets. The native, folded ABA structure has a nanopore unfolding time of 4.17 ms, compared with 0.29 ms for the ABA:ATP complex. A complementary 14‐mer strand, which binds the ABA sequence in the key nucleic acids responsible for folding, forms linear duplex DNA, resulting in a nanopore transit time of 0.50 ms and a higher capture probability than that of the folded ABA oligomer. Competition assays between the ABA:ATP and ABA:reporter complexes are carried out, and the results suggest that the ABA:ATP complex is formed preferentially. The nanopore allows for the detection of an ABA in its folded, ATP‐bound, and linear conformations.     

Single‐Molecule Manipulation in Zero‐Mode Waveguides

The mechanobiology of receptor–ligand interactions and force‐induced enzymatic turnover can be revealed by simultaneous measurements of force response and fluorescence. Investigations at physiologically relevant high labeled substrate concentrations require total internal reflection fluorescence microscopy or zero mode waveguides (ZMWs), which are difficult to combine with atomic force microscopy (AFM). A fully automatized workflow is established to manipulate single molecules inside ZMWs autonomously with noninvasive cantilever tip localization. A protein model system comprising a receptor–ligand pair of streptavidin blocked with a biotin‐tagged ligand is introduced. The ligand is pulled out of streptavidin by an AFM cantilever leaving the receptor vacant for reoccupation by freely diffusing fluorescently labeled biotin, which can be detected in single‐molecule fluorescence concurrently to study rebinding rates. This work illustrates the potential of the seamless fusion of these two powerful single‐molecule techniques.     

Controlling DNA Tug‐of‐War in a Dual Nanopore Device

Methods for reducing and directly controlling the speed of DNA through a nanopore are needed to enhance sensing performance for direct strand sequencing and detection/mapping of sequence‐specific features. A method is created for reducing and controlling the speed of DNA that uses two independently controllable nanopores operated with an active control logic. The pores are positioned sufficiently close to permit cocapture of a single DNA by both pores. Once cocapture occurs, control logic turns on constant competing voltages at the pores leading to a “tug‐of‐war” whereby opposing forces are applied to regions of the molecules threading through the pores. These forces exert both conformational and speed control over the cocaptured molecule, removing folds and reducing the translocation rate. When the voltages are tuned so that the electrophoretic force applied to both pores comes into balance, the life time of the tug‐of‐war state is limited purely by diffusive sliding of the DNA between the pores. A tug‐of‐war state is produced on 76.8% of molecules that are captured with a maximum two‐order of magnitude increase in average pore translocation time relative to the average time for single‐pore translocation. Moreover, the translocation slow‐down is quantified as a function of voltage tuning and it is shown that the slow‐down is well described by a first passage analysis for a 1D subdiffusive process. The ionic current of each nanopore provides an independent sensor that synchronously measures a different region of the same molecule, enabling sequential detection of physical labels, such as monostreptavidin tags. With advances in devices and control logic, future dual‐pore applications include genome mapping and enzyme‐free sequencing.     

Solid-State Nanopore Array: Manufacturing and Applications

Nanopore brings extraordinary properties for a variety of potential applications in various industrial sectors. Since manufacturing of solid-state nanopore is first reported in 2001, solid-state nanopore has become a hot topic in the recent years. An increasing number of manufacturing methods have been reported, with continuously decreased sizes from hundreds of nanometers at the beginning to ≈1 nm until recently. To enable more robust, sensitive, and reliable devices required by the industry, researchers have started to explore the possible methods to manufacture nanopore array which presents unprecedented challenges on the fabrication efficiency, accuracy and repeatability, applicable materials, and cost. As a result, the exploration of fabrication of nanopore array is still in the fledging period with various bottlenecks. In this article, a wide range of methods of manufacturing nanopores are summarized along with their achievable morphologies, sizes, inner structures for characterizing the main features, based on which the manufacturing of nanopore array is further addressed. To give a more specific idea on the potential applications of nanopore array, some representative practices are introduced such as DNA/RNA sequencing, energy conversion and storage, water desalination, nanosensors, nanoreactors, and dialysis.     

Single‐Molecule Electrical Biosensors Based on Single‐Walled Carbon Nanotubes

Interactions between biological molecules are fundamental to biology. Probing the complex behaviors of biological systems at the molecular level provides new opportunities to uncover the wealth of molecular information that is usually hidden in conventional ensemble experiments and address the “unanswerable” questions in the physical, chemical and biological sciences. Nanometer‐scale materials are particularly well matched with biomolecular interactions due to their biocompatibility, size comparability, and remarkable electrical properties, thus setting the basis for biological sensing with ultrahigh sensitivity. This brief review aims to highlight the recent progress of the burgeoning field of single‐molecule electrical biosensors based on nanomaterials, with a particular focus on single‐walled carbon nanotubes (SWNTs), for better understanding of the molecular structure, interacting dynamics, and molecular functions. The perspectives and key issues that will be critical to the success of next‐generation single‐molecule biosensors toward practical applications are also discussed, such as the device reproducibility, system integration, and theoretical simulation.     

用于DNA分子检测的纳米电极

基于纳米孔技术的新兴单分子传感技术已开发用于DNA测序,使得测序技术读取数据更快、测序成本降低、测序样品简化、无需标记.纳米孔孔内集成横向纳米电极可实现二维双通道同时检测易位信号的变化,从而提高纳米孔测序的精确度.目前纳米电极的制备方法主要包括:机械可控劈裂结法、电子束光刻法、电子束诱导沉积法、聚焦离子束刻蚀法、透射电镜刻蚀法等.分析纳米电极不同制备方法、应用范围、表征及其优缺点,可为后续纳米孔技术应用到DNA测序平台提供高效、稳定的选择.     

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.     

Nonadditive Transport in Multi‐Channel Single‐Molecule Circuits

As stated in the classic Kirchhoff's circuit laws, the total conductance of two parallel channels in an electronic circuit is the sum of the individual conductance. However, in molecular circuits, the quantum interference (QI) between the individual channels may lead to apparent invalidity of Kirchhoff's laws. Such an effect can be very significant in single‐molecule circuits consisting of partially overlapped multiple transport channels. Herein, an investigation on how the molecular circuit conductance correlates to the individual channels is conducted in the presence of QI. It is found that the conductance of multi‐channel circuit consisting of both constructive and destructive QI is significantly smaller than the addition of individual ones due to the interference between channels. In contrast, the circuit consisting of destructive QI channels exhibits an additive transport. These investigations provide a new cognition of transport mechanism and manipulation of transport in multi‐channel molecular circuits.