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.     

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.     

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.

     

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.