Nanopores: A journey towards DNA sequencing

Much more than ever, nucleic acids are recognized as key building blocks in many of life's processes, and the science of studying these molecular wonders at the single-molecule level is thriving. A new method of doing so has been introduced in the mid 1990's. This method is exceedingly simple: a nanoscale pore that spans across an impermeable thin membrane is placed between two chambers that contain an electrolyte, and voltage is applied across the membrane using two electrodes. These conditions lead to a steady stream of ion flow across the pore. Nucleic acid molecules in solution can be driven through the pore, and structural features of the biomolecules are observed as measurable changes in the trans-membrane ion current. In essence, a nanopore is a high-throughput ion microscope and a single-molecule force apparatus. Nanopores are taking center stage as a tool that promises to read a DNA sequence, and this promise has resulted in overwhelming academic, industrial, and national interest. Regardless of the fate of future nanopore applications, in the process of this 16-year-long exploration, many studies have validated the indispensability of nanopores in the toolkit of single-molecule biophysics. This review surveys past and current studies related to nucleic acid biophysics, and will hopefully provoke a discussion of immediate and future prospects for the field.     

Coordination-based gold nanoparticle layers

Gold nanoparticle (NP) mono- and multilayers were constructed on gold surfaces using coordination chemistry. Hydrophilic Au NPs (6.4 nm average core diameter), capped with a monolayer of 6-mercaptohexanol, were modified by partial substitution of bishydroxamic acid disulfide ligand molecules into their capping layer. A monolayer of the ligand-modified Au NPs was assembled via coordination with Zr4+ ions onto a semitransparent Au substrate (15 nm Au, evaporated on silanized glass and annealed) precoated with a self-assembled monolayer of the bishydroxamate disulfide ligand. Layer-by-layer construction of NP multilayers was achieved by alternate binding of Zr4+ ions and ligand-modified NPs onto the first NP layer. Characterization by atomic force microscopy (AFM), ellipsometry, wettability, transmission UV-vis spectroscopy, and cross-sectional transmission electron microscopy showed regular growth of NP layers, with a similar NP density in successive layers and gradually increased roughness. The use of coordination chemistry enables convenient step-by-step assembly of different ligand-possessing components to obtain elaborate structures. This is demonstrated by introducing nanometer-scale vertical spacing between a NP layer and the gold surface, using a coordination-based organic multilayer. Electrical characterization of the NP films was carried out using conductive AFM, emphasizing the barrier properties of the organic spacer multilayer. The results exhibit the potential of coordination self-assembly in achieving highly controlled composite nanostructures comprising molecules, NPs, and other ligand-derivatized components.     

Assembly of coordination nanostructures via ligand derivatization of oxide surfaces

A scheme is presented for the construction of coordination nanostructures on oxide surfaces (glass, Si/SiO2, quartz), based on application of epoxy-terminated monolayers as anchors for covalent grafting of ligands. Two ligands bearing amine groups were reacted with epoxysilane monolayers on oxide surfaces, providing ligand-terminated substrates. The ligands employed were (i) a pyridine moiety, used for subsequent binding of cobalt tetraphenylporphine (CoTPP), and (ii) deferoxamine (DFX), which contains hydroxamic acid moieties, used for subsequent construction of various Zr4+-based coordination layers. The results suggest that a dense ligand layer was obtained in both cases, allowing the formation of coordination overlayers on the oxide surfaces. The growth of coordinated layers was similar to analogous overlayers assembled on Au substrates, indicating that high ligand coverage is achieved by the epoxy-amine surface reaction. Epoxy-based functionalization of oxide substrates is a mild and efficient method for preparing high-quality coordination overlayers. Moreover, the method makes use of commercially available silane and amine reactants, providing the basis for wide application.     

Discrimination of methylcytosine from hydroxymethylcytosine in DNA molecules

Modified DNA bases are widespread in biology. 5-Methylcytosine (mC) is a predominant epigenetic marker in higher eukaryotes involved in gene regulation, development, aging, cancer, and disease. Recently, 5-hydroxymethylcytosine (hmC) was identified in mammalian brain tissue and stem cells. However, most of the currently available assays cannot distinguish mC from hmC in DNA fragments. We investigate here the physical properties of DNA with modified cytosines, in efforts to develop a physical tool that distinguishes mC from hmC in DNA fragments. Molecular dynamics simulations reveal that polar cytosine modifications affect internal base pair dynamics, while experimental evidence suggest a correlation between the modified cytosine's polarity, DNA flexibility, and duplex stability. On the basis of these physical differences, solid-state nanopores can rapidly discriminate among DNA fragments with mC or hmC modification by sampling a few hundred molecules in the solution. Further, the relative proportion of hmC in the sample can be determined from the electronic signature of the intact DNA fragment.     

Measurable versions of the LS category on laminations

We give two new versions of the LS category for the set-up of measurable laminations defined by Bermúdez. Both of these versions must be considered as “tangential categories”. The first one, simply called (LS) category, is the direct analogue for measurable laminations of the tangential category of (topological) laminations introduced by Colman Vale and Macías Virgós. For the measurable lamination that underlies any lamination, our measurable tangential category is a lower bound of the tangential category. The second version, called the Λ-category, depends on the choice of a transverse invariant measure Λ. We show that both of these “tangential categories” satisfy appropriate versions of some well known properties of the classical category: the homotopy invariance, existence of a dimensional upper bound, a cohomological lower bound (cup length), and an upper bound given by the critical points of a smooth function. Also, we show possible applications of these invariants to variational problems.     

Widely-applicable gold substrate for the study of ultrathin overlayers

Ultrathin films on gold substrates have been the subject of enormous scientific and technological interest. Comprehensive study of such systems requires concomitant application of a variety of complementary characterization techniques. The reliability of the result is frequently hampered by the fact that different characterization methods impose different requirements on the Au substrate, resulting in the need to use different types of Au substrates for different measurements, possibly influencing the overlayer structure. This results in an average, rather than exact, structure determination. Here, we show that 15-nm-thick Au films evaporated at 0.5 A/sec on silanized glass and annealed are semi-transparent, electrically conducting, and morphologically well-defined, showing a smooth, [111] textured surface. Such Au films provide a high-quality, widely applicable and relatively inexpensive platform for ultrathin overlayers, enabling characterization by a wide spectrum of experimental methods, applied to the same substrate. The exceptional qualities and analytical capabilities of such substrates are demonstrated with several different systems: (i) Cu underpotential deposition (upd); (ii) alkanethiol self-assembly; (iii) formation of Au nanoparticle layers; (iv) binding of the chromophore protoporphyrin IX (PPIX) to a monolayer of 11-mercaptoundecanoic acid (MUA). In the latter case it is shown that the use of Cu(2+) ions for binding between the carboxylate groups of PPIX and MUA promotes better organization of the porphyrin layer.     

Branched coordination multilayers on gold

A C3-symmetric tridentate hexahydroxamate ligand molecule was specially synthesized and used for coordination self-assembly of branched multilayers on Au surfaces precoated with a self-assembled monolayer (SAM) of ligand anchors. Layer-by-layer (LbL) growth of multilayers via metal-organic coordination using Zr4+ ions proceeds with high regularity, adding one molecular layer in each step, as shown by ellipsometry, wettability, UV-vis spectroscopy, and atomic force microscopy (AFM). The branched multilayer films display improved stiffness, as well as a unique defect self-repair capability, attributed to cross-linking in the layers and lateral expansion over defects during multilayer growth. Transmetalation, i.e., exposure of Zr4+-based assemblies to Hf4+ ions, was used to evaluate the cross-linking. Conductive atomic force microscopy (AFM) was used to probe the electrical properties of the multilayers, revealing excellent dielectric behavior. The special properties of the branched layers were emphasized by comparison with analogous multilayers prepared similarly using linear (tetrahydroxamate) ligand molecules. The process of defect annihilation by bridging over defective areas, attributed to lateral expansion via the excess bishydroxamate groups, was demonstrated by introduction of artificial defects in the anchor monolayer, followed by assembly of two layers of either the linear or the branched molecule. Analysis of selective binding of Au nanoparticles (NPs) to unblocked defects emphasized the superior repair mechanism in the branched layers with respect to the linear ones.     

A rapid approach to reproducible, atomically flat gold films on mica

A simple and reproducible method for the preparation of gold films on mica with large (typically 0.8-1.0 μm) atomically flat (1 1 1) terraces is described. The procedure involves thin gold film evaporation onto freshly cleaved mica substrates, followed by 1 min annealing at 650 °C under nitrogen. The annealed gold surfaces are compared to those of freshly evaporated gold films on mica using cyclic voltammetry and atomic force microscopy. Our results favorably compare to other published annealing techniques, with minimal equipment and time necessary to reproducibly obtain atomically flat gold terraces.     

Divergent growth of coordination dendrimers on surfaces

Divergent growth of surface-initiated dendritic nanostructures on gold surfaces in a highly controlled, stepwise manner is demonstrated, using metal-organic coordination as the binding motif. The repeat unit for dendrimer growth was a branched, C3-symmetrical ligand building block bearing three bis-hydroxamate groups. The surface initiation sites for dendrimer growth were obtained by the formation of a mixed monolayer comprising isolated bis-hydroxamate disulfide anchor ligands and octanethiol (OT) at very low anchor/OT ratios. Following functionalization of the surface with spaced anchors, alternate immersion in solutions of Zr4+ ions and the branched ligand afforded surface-confined dendrimers of increasing generation, where the number of generations is conveniently controlled by the number of coordination binding sequences. The heights of different generation dendrimers are in excellent agreement with values predicted by molecular models, as well as with thicknesses of branched multilayers prepared by the same procedure on full anchor monolayers. At higher generation numbers, gradual dendrimer overlap and coalescence are observed, eventually resulting in a continuous overlayer and a transition from 3D to 1D growth. A mechanism for the development of dendritic coordination nanostructures on surfaces is discussed.