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Nanopore Basics

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Like most great ideas [1,2], the idea behind nanopores probably began with a crudely scribbled diagram on a bar napkin: if single-stranded DNA could be forced through a tiny hole in a membrane that was passing electrical current, then the blockage caused by each base could be used to directly read the sequence. While the practical implementation of this idea turned out to be much more complicated than initially envisioned, from these humble beginnings was born an entire field of multidisciplinary research that today comprises thousands of researchers, hundreds of laboratories worldwide, and the beginnings of commercial enterprise. And now, after almost 30 years, the idea actually works [3].

Nanopores are useful for far more than just DNA sequencing, however. The basic premise of using nanopores to interrogate individual biomolecules is very simple. An insulating membrane containing a nanometre-scale pore is immersed between two fluidic reservoirs containing electrolyte solution, such that the only connection between the two reservoirs is through the pore. When a potential difference is applied across the membrane, ionic current is driven through the pore. If charged biomolecules are also present in the solution, they are driven through the pore as well, producing transient current blockades from which information about the biomolecule can be extracted.

There are two major classes of nanopores. The first consists of biological protein pores derived from cell membrane proteins. These pores exist in every cell in every living thing on the planet and are used in vivo for an astonishing variety of purposes, including ion pumps for chemical energy generation; maintaining electrolyte imbalances for nerve signalling; mediating selective biomolecular transport through cell membranes; and much more. From among the near-infinite variety of wild-type pores available, two in particular have come to be widely used for research purposes: alpha-hemolysin (aHL) [4], a pore derived from the toxin Staphylococcus aureus, and Mycobacterium smegmatis porin A (MspA) [5]. The first DNA translocation experiments were performed using aHL pores. Currently, the MinION sequencer available from Oxford Nanopore uses a heavily engineered mutant of the CsgG pore, an amyloid secretion channel found in Escherichia coli, to achieve long read sequencing [6].

The second class of nanopore are solid-state pores in thin synthetic membranes [7,8]. These are quite literally tiny holes in thin membranes. Unlike their biological counterparts, solid-state nanopores are robust, flexible, and can relatively easily be integrated into other solid-state electronic devices, making them preferable as a long-term solution over biological pores if the shortcomings of their comparatively lower sensitivity can be addressed. On the other hand, solid-state nanopores have yet to achieve the level of motion control required for reliable interpretation of translocation signals of complex biopolymers, though impressive work is being done with synthetic polymers in the information storage space [9,10].

Solid-state nanopores are divided into a few subclasses. Planar pores, which are NNi’s focus, are the ones we have discussed so far and are by far the most commonly used type. The main alternative is nanopipettes, thin glass capillaries extended to a nanoscale opening. In practice they operate very similarly to solid-state nanopores, having longer effective lengths and consequently reduced spatial sensitivity, but lower noise due to the glass substrate.

At NNi, we believe that solid-state nanopores are the future of the nanopore field, and so we will focus this course on solid-state nanopores. Most of this course applies equally well to any type of nanopore and we are more than happy to support biological pore research however we can.


[1] D. Deamer, M. Akeson, and D. Branton, “Three decades of nanopore sequencing,” Nat. Biotechnol., vol. 34, no. 5, pp. 518–524, 2016. doi:

[2] J. J. Kasianowicz and S. M. Bezrukov, “On `three decades of nanopore sequencing’ Reply,” Nat. Biotechnol., vol. 34, no. 5, p. 482, 2016.

[3] C. G. Brown and J. Clarke, “Nanopore development at Oxford Nanopore,” Nat. Biotechnol., vol. 34, p. 810, Aug. 2016.

[4] J. J. Kasianowicz, E. Brandin, D. Branton, and D. W. Deamer, “Characterization of individual polynucleotide molecules using a membrane channel,” Proc. Natl. Acad. Sci. U.S.A., vol. 93, no. November, p. 13770, 1996.

[5] E. A. Manrao et al., “Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.,” Nat. Biotechnol., vol. 30, no. 4, pp. 349–53, Apr. 2012.

[6] J.-M. Carter and S. Hussain, “Robust long-read native DNA sequencing using the ONT CsgG Nanopore system,” Wellcome Open Res., vol. 2, no. May, p. 23, 2017.

[7] H. Kwok, K. Briggs, and V. Tabard-Cossa, “Nanopore fabrication by controlled dielectric breakdown.,” PLoS One, vol. 9, no. 3, p. e92880, Jan. 2014.

[8] J. Li, D. Stein, C. McMullan, D. Branton, M. J. Aziz, and J. A. Golovchenko, “Ion-beam sculpting at nanometre length scales,” Nature, vol. 412, no. 6843, pp. 166–169, 2001.

[9] K. Chen, J. Kong, J. Zhu, N. Ermann, P. Predki, and U. F. Keyser, “Digital Data Storage Using DNA Nanostructures and Solid-State Nanopores,” Nano Lett., vol. 19, pp. 1210–1215, 2019.

[10] R. Lopez et al., “DNA assembly for nanopore data storage readout,” Nat. Commun., vol. 10, no. 1, pp. 1–9, 2019.

Last Updated: 2022-11-10


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