How to make a solid-state nanopore


Until recently, planar solid-state nanopores (ssNP) were typically fabricated using a transmission electron microscope or ion beam microscope [1,2], by focusing the beam down to a nanoscale spot and literally drilling a hole. This practice sustained the field for the better part of 20 years. This is a testament to the persistence and dedication of early solid-state nanopore researchers, since these methods are expensive, hard to master, and even highly skilled operators regularly fail to make a useful pore at the end of a long and arduous process. The pores are made dry, and many are lost to the handling steps needed to get them wetted and ready for sensing. While you do get an image of your nanopore for free while making it, a major issue that you will not find discussed in the literature is that the pore that is left when you have finished cleaning and wetting the chip is usually quite different from the pore that you saw in the TEM. Finally, the requirement of a beam-drilling tool has until now limited nanopore research to a few well-established research institutions, excluding an enormous number of highly talented researchers from taking part in this exciting field of study.


In 2014 we published the controlled breakdown (CBD) method of nanopore fabrication [3], which does away with all of the expensive equipment required to fabricate and use solid-state nanopores and finally makes the technology broadly accessible. The idea is essentially to harness a static shock to do the work for us: by applying voltage near the dielectric strength of the material across an intact membrane, we drive dielectric breakdown, the same physical processes that causes static shocks in air. On the nano scale and in the solid state, however, this spark will leave behind a nanopore on the scale of 1 nm [4], which can then be enlarged to any desired size in the same setup [5]. This section goes into detail on how to make nanopores in silicon nitride membranes with NNi equipment using the controlled breakdown method.

One of the many benefits of the CBD method is that it is universal, able to fabricate a nanopore in any dielectric membrane [6], including 2D materials like graphene [7] and TDMCs [8], and even through membrane stacks involving conductive layers [9]. If your research requires optimization for a different membrane material, get in touch and we will be more than happy to assist you in optimizing protocols for your specific requirements.


Cleaning the Membrane

Required Materials:

  • 30 mL sulfuric acid

  • 10 mL hydrogen peroxide

  • NNi Piranha cleaning jig

  • 100 mL beaker

  • Hot plate

  • 1 glass pipettes

  • Acid waste container

  • Acid-compatible PPE (apron, face shield, long gloves)

  • Access to a fume hood

The first consideration is membrane cleanliness. Membranes stored in gel packs will accumulate a layer of hydrocarbons from the gel which render the membrane hydrophobic and difficult to wet, which will in turn promote nanobubbles and noisy pores. A clean surface is critical, and is best achieved using a Piranha clean, which is detailed in a recent Nature Protocols publication [10]. A tutorial will be coming soon.


Note that Piranha, as the name suggests, is extremely dangerous if not handled with care. Proper protective equipment and training are required to use it safely. NNi carries a line of jigs built to facilitate Piranha cleaning, which we strongly recommend as a means to simplify the handling process. This jig is included in our standard lab starter pack.


Mounting the membrane

Required Materials:

  • 2 NNi Flow Cell sets

  • Tweezers

  • 1 mL NNi Fabrication Buffer

  • Access to a 100uL pipette

  • 2 recently-cleaned SiNx membranes

Once the chip has been cleaned, it’s time to mount it in your flow cell and wet the chip with NNi Fabrication Buffer. A detailed set of protocols for doing so can be found here.

NNi sells two version of flow cells. The first set are fully disposable, intended to be used once and discarded without ever dismounting the membrane. This avoids cross-contamination between experiments and simplifies and speeds up both experimental preparation and cleanup. The second version of the flow cell employs a male half-cell that can be reused a few times. An assembled cell can easily be disassembled by pulling the two halves apart gently, taking care to pull straight as you do so to avoid snapping the clips.


When considering which flow cell is right for you, the main consideration is cross-contamination between experiments. This is rarely a problem for simple studies involving nucleic acids, which do not stick to the flow cells in significant quantities and are easily cleaned simply by flushing out the buffer. For these studies, NNi recommends reusable flow cells and simply flushing out the flow cell with dionized water between experiments. For protein studies, or studies with more exotic synthetic molecules, care should be taken to ensure that sample can be effectively flushed out between experiments before reusing half cells. By default, if using a target molecule other than DNA, as strongly recommend using disposable flow cells.


Note that the half cells are not compatible with acids or most organic solvents, so intense cleaning protocols are not recommended. Flow cells are sufficiently inexpensive that if there is any doubt, it is usually cost-effective to simply use a new half-cell as compared to taking lots of time to clean in any case.


Fabricating the pore

Required Materials:

  • 2 mounted membranes in NNi flow cells

  • Spark-E2

  • 1mL NNi Conditioning Buffer

  • Access to a 100uL pipette

  • Access to NNi Nanopore Fabrication Software

Fabricating the nanopore with NNi’s tools is very simple. Mount the flow cells in the Spark-E2 unit, select your target pore size, and press go.



By default, the protocol that ships with the software is optimized for a 3-10 nm diameter nanopore in a 10-12 nm thick SiNx membrane, and will generally achieve your target size within 0.5 nm most of the time in that range. The video here goes into some detail demonstrating the use of the software.


Some small tweaks to the protocol are needed if you need a very small and precise nanopore, or if you are targeting a very large pore. We are happy to provide optimized protocols for your specific pore requirements on demand, and we encourage you to reach out to us for a quick consultation if the standard protocols are not meeting your specifications or if there are any other customization needed to enable your research.


Table of Contents

Previous Topic: Nanopore basics

Next Topic: Characterizing a nanopore electrically


References

[1] 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, https://doi.org/10.1038/35084037.


[2] A. J. Storm, J. H. Chen, X. S. Ling, H. W. Zandbergen, and C. Dekker, “Fabrication of solid-state nanopores with single-nanometre precision,” Nat. Mater., vol. 2, p. 537, 2003, https://doi.org/10.1038/nmat941.


[3] H. Kwok, K. Briggs, and V. Tabard-Cossa, “Nanopore fabrication by controlled dielectric breakdown.,” PLoS One, vol. 9, no. 3, p. e92880, 2014, https://doi.org/10.1371/journal.pone.0092880.


[4] K. Briggs, H. Kwok, and V. Tabard-Cossa, “Automated fabrication of 2-nm solid-state nanopores for nucleic acid analysis,” Small, vol. 10, no. 10, 2014, https://doi.org/10.1002/smll.201303602.


[5] C. Leung, K. Briggs, M.-P. Laberge, S. Peng, M. Waugh, and V. Tabard-Cossa, “Mechanisms of solid-state nanopore enlargement under electrical stress,” Nanotechnology, vol. 31, no. 44, p. 44LT01, 2020, https://doi.org/10.1088/1361-6528/aba86e


[6] J. Larkin, R. Henley, D. C. Bell, T. Cohen-Karni, J. K. Rosenstein, and M. Wanunu, “Slow DNA Transport through Nanopores in Hafnium Oxide Membranes,” ACS Nano, vol. 7, no. 11, pp. 10121--10128, 2013, https://doi.org/10.1021/nn404326f.


[7] A. T. Kuan, B. Lu, P. Xie, T. Szalay, and J. A. Golovchenko, “Electrical pulse fabrication of graphene nanopores in electrolyte solution,” Appl. Phys. Lett., vol. 106, no. May, p. 203109, 2015, https://doi.org/10.1063/1.4921620.


[8] J. Feng et al., “Electrochemical reaction in single layer MoS2: Nanopores opened atom by atom,” Nano Lett., vol. 15, no. 5, pp. 3431–3438, 2015, https://doi.org/10.1021/acs.nanolett.5b00768.


[9] H. Kwok, M. Waugh, J. Bustamante, K. Briggs, and V. Tabard-Cossa, “Long passage times of short ssDNA molecules through metallized nanopores fabricated by controlled breakdown,” Adv. Funct. Mater., vol. 24, no. 48, 2014, https://doi.org/10.1002/adfm.201402468.


[10] M. Waugh et al., “Solid-state nanopore fabrication by automated controlled breakdown,” Nat. Protoc., vol. 15, pp. 122–143, 2019, https://doi.org/10.1038/s41596-019-0255-2.


Last Updated: 2021-05-11