In a world dominated by computers it is still a challenge for electronics to interact with the molecules that control our bodies, cause disease and make up important industrial chemicals. Nanopores allow a simple electronic system to measure DNA molecules, count viruses and detect proteins. A nanopore sensor is a hole in a membrane with a diameter as small as 1- 2 nanometres, combined with a means of electronically detecting single molecules passing through the hole. Nanopore sensors have the potential to be used in areas as diverse as healthcare, computing and industry.
DNA sequencing has been the greatest driver of progress in nanopore sensing over the last three decades. Oxford Nanopore’s MinION device is now available in an extremely small form factor1. However, the obvious progress made in DNA sequencing shouldn’t distract us from the other opportunities presented by this technology; Nanopores can also be used to detect other biomolecules and many polymers.
The resistive pulse sensing technique used in nanopore sensors is well established. It’s used in Coulter Counters in hospitals to count red blood cells as they pass through a small hole in a membrane between two reservoirs. Every time a blood cell passes through the hole it blocks the electrical current that is otherwise flowing through the hole. By measuring how this current is reduced you can count the number of cells and their size. A nanopore sensor works in a similar way but the diameter of the pore is reduced by 10,000 times to be just a few nanometres across. This is close to the size of single strands of DNA. As each molecule blocks the pore the change in current can be used to help characterise it.
It is challenging to accurately fabricate such small pores and control the translocation of molecules through them. There are two routes to creating nanopores. The first is to exploit the self-assembly of biological molecules, such as proteins. The second route is to define pores using the top down processing techniques developed for making computer processors in silicon – solid state pores. One method is to use the high energy electron beam in a transmission electron microscope to drill a hole. This is a slow process and recently it has been demonstrated that high voltages in liquids can be used to form a pore in commonly used silicon nitride membranes. This allows nanometre sized pores to be created much more quickly2.
Whilst there are significant challenges with sequencing DNA through solid state pores, there are a number of more tractable, diagnostic applications worth considering:
- By binding probes to known sequences of DNA it is possible to determine the presence of single nucleotide polymorphisms which have important diagnostics applications3.
- Slightly larger pores, with a diameter of 100 – 500 nm, can be used to count viruses. In these cases, the key advantages of using nanopores include very low sample volumes, not requiring labeling and the potential to make measurements of unknown targets4.
- Nanopores are already in use in research to study protein unfolding, investigate DNA protein binding and even measure protein activity5.
Industry and DNA Computing
Although much nanopore development has focused on biomolecules this has often been driven by ease of use in this application and funding priorities. Because of this, it is likely that industrial targets for nanopore sensing have been overlooked. For example, nanopores can also be used to provide a real-time measurement, akin to mass spectrometry, of polymer solutions6.
Combining nanopore sensors with DNA nanotechnology and biochemistry opens up even more opportunities. For example, strands of DNA have been folded to create barcoded structures that also bind to specific proteins7. This enables digital multiplexed measurements of protein concentration in a sample. These barcodes also represent a way of storing information on DNA in a way that is easier to read and write than directly storing it in the sequence. Data stored on DNA would have incredibly high data density and very high stability.
Using nanopores to read information encoded on single molecules could also be used to interpret the results of molecular computers. Calculations made by molecular reactions have the potential to be extremely parallel and low power. However, it can be hard to determine the output, limiting the complexity of the problems they can be used to solve. If the result of the calculation is encoded on a DNA molecule then perhaps a nanopore sensor could quickly read this result and assess its concentration.
This post has highlighted just a few of the long list of different measurements that can be made using nanopore sensors. Desktop nanopore devices, no larger than a portable USB hard drive have already been demonstrated allowing single molecule measurements to made outside of the lab. DNA sequencing companies are interested in the potential of nanopores and there are a number of start-ups exploring this technology for other applications. We can expect these versatile sensors to be used in many settings for many different uses.
Michael is a Bioinstrumentation Consultant at TTP. He has completed a PhD and Post Doc in the Cavendish Laboratory, University of Cambridge on graphene membranes for nanopore sensing and filtration. He has experience fabricating nanopores in a number of different materials and then using them to detect single molecules. His background is in electronic engineering and his interest is in combining electronic systems with bio-physics to create systems to interpret and manipulate biological molecules.
- Oxford Nanpore https://nanoporetech.com/products/minion
- Kwok et al, (2014) Nanopore Fabrication by Controlled Dielectric Breakdown. PLOS one. 9 (3) http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092880
- Kong et al, (2017). Chemical Communications. 2 http://pubs.rsc.org/en/content/articlelanding/2017/cc/c6cc08621g?iscitedby=True#!divAbstract
- Yang et al, (2016). Quantification of Virus Particles Using Nanopore-Based Resistive-Pulse Sensing Techniques. Front. Microbiol. 7 (1500) http://journal.frontiersin.org/article/10.3389/fmicb.2016.01500/full
- Zhou et al, (2016). Label-Free Nanopore Single-Molecule Measurement of Trypsin Activity. ACS Sens, 1 (5) http://pubs.acs.org/doi/abs/10.1021/acssensors.6b00043
- Reiner et al, (2010). Theory for polymer analysis using nanopore-based single-molecule mass. PNAS. 107 (27) spectrometry http://www.pnas.org/content/104/20/8207.abstract?ijkey=b5daec7bc9f449b9fe92150d385d5e982fb16ea0&keytype2=tf_ipsecsha
- Bell et al, (2016). Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores. Nature Nanotechnology. 11 http://www.nature.com/nnano/journal/v11/n7/abs/nnano.2016.50.html