The emergence of nanopores in next-generation sequencing.

Next-generation sequencing methods based on nanopore technology have recently gained considerable attention, mainly because they promise affordable and fast genome sequencing by providing long read lengths (5 kbp) and do not require additional DNA amplification or enzymatic incorporation of modified nucleotides. This permits health care providers and research facilities to decode a genome within hours for less than $1000. This review summarizes past, present, and future DNA sequencing techniques, which are realized by nanopore approaches such as those pursued by Oxford Nanopore Technologies.

Probing the size of proteins with glass nanopores.

Single molecule studies using nanopores have gained attention due to the ability to sense single molecules in aqueous solution without the need to label them. In this study, short DNA molecules and proteins were detected with glass nanopores, whose sensitivity was enhanced by electron reshaping which decreased the nanopore diameter and created geometries with a reduced sensing length. Further, proteins having molecular weights (MW) ranging from 12 kDa to 480 kDa were detected, which showed that their corresponding current peak amplitude changes according to their MW. In the case of the 12 kDa ComEA protein, its DNA-binding properties to an 800 bp long DNA molecule was investigated. Moreover, the influence of the pH on the charge of the protein was demonstrated by showing a change in the translocation direction. This work emphasizes the wide spectrum of detectable molecules using nanopores from glass nanocapillaries, which stand out because of their inexpensive, lithography-free, and rapid manufacturing process.

Controllable shrinking and shaping of glass nanocapillaries under electron irradiation.

The ability to reshape nanopores and observe their shrinkage under an electron microscope is a powerful and novel technique. It increases the sensitivity of the resistive pulse sensing and enables to detect very short and small molecules. However, this has not yet been shown for glass nanocapillaries. In contrast to their solid-state nanopore counterparts, nanocapillaries are cheap, easily fabricated and in the production do not necessitate clean room facilities. We show for the first time that quartz nanocapillaries can be shrunken under a scanning electron microscope beam. Since the shrinking is caused by the thermal heating of the electrons, increasing the beam current increases the shrink rate. Higher acceleration voltage on the contrary increases the electron penetration depth and reduces the electron density causing slower shrinkage. This allows us to fine control the shrink rate and to stop the shrinking process at any desired diameter. We show that a shrunken nanocapillary detects DNA translocation with six times higher signal amplitudes than an unmodified nanocapillary. This will open a new path to detect small and short molecules such as proteins or RNA with nanocapillaries.

Fast and automatic processing of multi-level events in nanopore translocation experiments.

We have developed a method to analyze in detail, translocation events providing a novel and flexible tool for data analysis of nanopore experiments. Our program, called OpenNanopore, is based on the cumulative sums algorithm (CUSUM algorithm). This algorithm is an abrupt change detection algorithm that provides fitting of current blockages, allowing the user to easily identify the different levels in each event. Our method detects events using adaptive thresholds that adapt to low-frequency variations in the baseline. After event identification, our method uses the CUSUM algorithm to fit the levels inside every event and automatically extracts their time and amplitude information. This facilitates the statistical analysis of an event population with a given number of levels. The obtained information improves the interpretation of interactions between the molecule and nanopore. Since our program does not require any prior information about the analyzed molecules, novel molecule-nanopore interactions can be characterized. In addition our program is very fast and stable. With the progress in fabrication and control of the translocation speed, in the near future, our program could be useful in identification of the different bases of DNA.

Note: Direct force and ionic-current measurements on DNA in a nanocapillary.

We have developed optical tweezers, with force measurements based on fast video tracking, for analysis and control of DNA translocation through nanocapillaries. Nanocapillaries are single-molecule biosensors with very similar characteristics to solid-state nanopores. Our novel experimental setup allows for ionic-current measurements in which the nanocapillary is oriented perpendicular to the trapping laser. Using video-based particle tracking, we are able to measure the position of DNA coated colloids at sub-millisecond resolution and in real-time. We present the first electrophoretic force and simultaneous ionic-current measurements of a single DNA molecule inside the orifice of a nanocapillary.

Probing DNA with micro- and nanocapillaries and optical tweezers.

We combine for the first time optical tweezer experiments with the resistive pulse technique based on capillaries. Quartz glass capillaries are pulled into a conical shape with tip diameters as small as 27 nm. Here, we discuss the translocation of λ-phage DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ-phage DNA molecules. Our flow cell design allows for the straightforward incorporation of optical tweezers. We show that a DNA molecule attached to an optically trapped colloid is pulled into a capillary by electrophoretic forces. The detected electrophoretic force is in good agreement with measurements in solid-state nanopores.

Sensing DNA-coatings of microparticles using micropipettes.

The resistive pulse technique is widely used to detect the size of small particles in aqueous solutions. This work demonstrates that a few tens of DNA molecules and thus the charges on a particle can be simply detected by pressure-driven translocation through a microcapillary based Coulter counter. The typical opening of the capillaries ranges from 2 to 6 microm. The custom-built system gives optical access using a high numerical aperture objective allowing to observe colloids passing the sensing volume by optical means. We show the feasibility of our setup by distinguishing colloids with one and two micron diameters. Our measurements prove that a few ten strands of DNA bound to the colloids can be detected. This can be achieved by simple comparison of current amplitudes for blank and coated colloids at low salt concentrations (2-40 mmol [NaCl]). Our results clearly demonstrate that the Coulter counter can be used to detect the surface charges on colloids. Moreover, the results are in good agreement with a dynamical computer model taking into account the full geometry of the capillary.