Solid-state nanopore analysis of human genomic DNA shows unaltered global 5-hydroxymethylcytosine content associated with early-stage breast cancer.

5-Hydroxymethylcytosine (5hmC), the first oxidized form of the well-known epigenetic modification 5-methylcytosine, is an independent regulator of gene expression and therefore a potential marker for disease. Here, we report on methods developed for a selective solid-state nanopore assay that enable direct analysis of global 5hmC content in human tissue. We first describe protocols for preparing genomic DNA derived from both healthy breast tissue and stage 1 breast tumor tissue and then use our approach to probe the net abundance of the modified base in each cohort. Then, we employ empirical data to adjust for the impact of nanopore diameter on the quantification. Correcting for variations in nanopore diameter among the devices used for analysis reveals no detectable difference in global 5hmC content between healthy and tumor tissue. These results suggest that 5hmC changes may not be associated with early-stage breast cancer and instead are a downstream consequence of the disease.

Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection.

The biotin-streptavidin bond is the strongest noncovalent bond in nature and is thus used extensively in biotechnology applications. However, the difficulty of releasing the bond without high temperatures or corrosive solutions can be a barrier to applications involving nucleic acids and other delicate substrates. Here, room-temperature phenol is employed to release biotin-tagged DNA constructs from streptavidin rapidly and efficiently. It is demonstrated that synthetic biotinylated DNA can be recovered at yields approaching 100% from both solution-phase and bead-bound streptavidin with as little as 12% (v/v) phenol, leaving the biotin tag active and reusable after extraction. As an application of this recovery method, biotinylated DNA fragments are isolated from a mixed solution to provide selectivity for solid-state nanopore detection.

Detecting and identifying small molecules in a nanopore flux capacitor.

A new method of molecular detection in a metallic-semiconductor nanopore was developed and evaluated with experimental and computational methods. Measurements were made of the charging potential of the electrical double layer (EDL) capacitance as charge-carrying small molecules translocated the nanopore. Signals in the charging potential were found to be correlated to the physical properties of analyte molecules. From the measured signals, we were able to distinguish molecules with different valence charge or similar valence charge but different size. The relative magnitude of the signals from different analytes was consistent over a wide range of experimental conditions, suggesting that the detected signals are likely due to single molecules. Computational modeling of the nanopore system indicated that the double layer potential signal may be described in terms of disruption of the EDL structure due to the size and charge of the analyte molecule, in agreement with Huckel and Debye's analysis of the electrical atmosphere of electrolyte solutions.

Active current gating in electrically biased conical nanopores.

We observed that the ionic current through a gold/silicon nitride (Si3N4) nanopore could be modulated and gated by electrically biasing the gold layer. Rather than employing chemical modification to alter device behavior, we achieved control of conductance directly by electrically biasing the gold portion of the nanopore. By stepping through a range of bias potentials under a constant trans-pore electric field, we observed a gating phenomenon in the trans-pore current response in a variety of solutions including potassium chloride (KCl), sodium chloride (NaCl), and potassium iodide (KI). A computational model with a conical nanopore was developed to examine the effect of the Gouy-Chapman-Stern electrical double layer along with nanopore geometry, work function potentials, and applied electrical bias on the ionic current. The numerical results indicated that the observed modulation and gating behavior was due to dynamic reorganization of the electrical double layer in response to changes in the electrical bias. Specifically, in the conducting state, the nanopore conductance (both numerical and experimental) is linearly proportional to the applied bias due to accumulation of charge in the diffuse layer. The gating effect occurs due to the asymmetric charge distribution in the fluid induced by the distribution of potentials at the nanopore surface. Time dependent changes in current due to restructuring of the electrical double layer occur when the electrostatic bias is instantaneously changed. The nanopore device demonstrates direct external control over nanopore behavior via modulation of the electrical double layer by electrostatic biasing.

The effects of the electrical double layer on giant ionic currents through single-walled carbon nanotubes.

We developed a computational model for investigating the cause for the high ionic current through a single-walled carbon nanotube nanofluidic device by considering the electrical double layer at a solid-liquid interface. With this model, we were able to examine the influence of the Gouy-Chapman-Stern electrical double layer and the solution concentration on the ionic conductance in the device. Results showed that the conductance-concentration relationship predicted from our model agreed well with experimental observation. Moreover, our model showed that the compact layer thickness increased with the increase of the bulk solution concentration, reducing the internal volume of the nanotube channel available for fluid transport. Fluid within the channel had an enhanced concentration and a net charge which increased the electroosmotic and electrophoretic transport properties of the device, increasing the total ionic conductance of the system.