The Fascinating World of Nanopores: DNA Sequencing and Beyond

We often hear the term "it's in our DNA", but what does this mean? How do we establish the characteristics of our DNA? Well, by reading it. DNA is a complex molecule that carries the genetic instructions for all living organisms. It is a double helix structure made up of nucleotides with four bases (A, T, C, G). By detecting and reading the order these bases appear in we can understand the genetic code that carries instructions for an organism's development and function. Various methods of DNA sequencing (i.e. reading the order the bases appear in) have been developed in the past half-century, but one has stood out in the last decade: nanopore sequencing. Nanopore sequencing revolutionised the world of DNA sequencing. What makes nanopores stand out over long-established technologies is their ability to analyse one molecule at a time, in real-time, without the need to prior modify its natural state (Lin et al., 2021).

The power of nanopores does not stop here. Understanding the complexity of how living organisms function cannot rely on deciphering just the DNA; it depends upon going further, to analyse other molecules that constitute it (i.e., biomolecules). Only looking at the DNA is similar to deciding if a cake is tasty by looking only at the recipe. Fortunately, the capabilities of nanopores cover numerous types of biomolecules (including DNA, RNA, proteins, and small molecules) (Lin et al., 2021), which, using the cake analogy, would relate to not only reading the recipe, but also tasting the individual ingredients, and the final product.

All of these qualities allow for applications in research, medicine, and environmental monitoring (Tanimoto et al., 2022), to name a few. To paint a complete picture of the broad spectrum of nanopore potential, firstly the working principle will be explained, followed by highlighting the benefits of using nanopores and finally giving hands-on examples of the applicability of this potential in several fields.

Figure 1- 3D representation of a biological nanopore (ClyA) embedded in a lipid bilayer used for single-protein fingerprinting (Strack, 2022).

The Fundamentals of Nanopores

In essence, the experimental arrangement involves two compartments containing a liquid through which electrical current can flow. These are separated by a barrier that does not allow any exchange between them. A hole (of nanometres dimensions [10-9m]) referred to as the nanopore is present in this barrier and offers an exchange channel between the compartments (Fan et al., 2021). By applying a slight electric current through the nanopore the molecules present in the liquid move from one compartment to the other; the nanopore being the only available route. Because the nanopore is only slightly bigger than the molecules it is possible for only one molecule to interact with and pass through the nanopore at a time. The electric current flowing between the compartments is constantly measured and naturally, when there is a molecule present in the nanopore, there are changes in the current. The modifications in the electrical signal that result from this process can be interpreted to infer the properties or activities of the molecule (Tabard-Cossa, 2013). This process is called nanopore sensing.

Nanopore sensing devices can be divided into biological and solid-state categories. Each category has distinct advantages and drawbacks and is used for different applications. In the case of biological nanopores, the hole is a pore-forming protein and the barrier is formed by a double layer of lipids i.e., oils or any compound that is not soluble in water. Lipids are generally known as nutrients but here they serve a different purpose, to prevent communication between the two compartments and hold the pore. This double layer is also known as a lipid bilayer (Bhatti et al., 2021). They are better studied and characterised, and are more suitable for a laboratory environment. In solid-state nanopores the barrier is made using various materials (e.g., different types of silicones) and the tiny hole is poked in the barrier using different fabrication techniques (Gibb & Ayub, 2013). These offer more portability making them suitable for various, on-field environments.

Figure 2 - Schematical representation of biological and solid-state nanopores (Reynaud et al., 2020)

How Did Nanopores Revolutionise Biomolecule Analysis?

By enabling single-molecule detection and analysis, in a real-time and label-free manner, nanopores have revolutionised molecule sensing. Single-molecule detection entails sensing and analysing individual molecules as they pass through the nanopore. This is possible due to the size of the nanopore which is only slightly larger than that of the molecule to be analysed, meaning only one molecule can pass through the nanopore at a time. Hence, it is possible to study and understand each molecule on its own, which was not possible with traditional equipment (Ying & Long, 2019). Moreover, in contrast to traditional methods that require labelled molecules for detection, nanopores operate in a label-free manner, eliminating the need for prior modifications of the molecule being analysed, hence it remains in its natural state. To provide a real-world parallel, nanopores would be able to tell the name of a new person without them wearing a name tag. This avoids potential interference with the molecule's natural function, reduces labour-intensive steps, and lowers overall costs (Ying et al., 2022).

Figure 3 - In 2014, In 2014, Oxford Nanopore Technologies (ONT) introduced MinION, the inaugural nanopore sequencer. This ground-breaking technology operates by utilising a miniature protein pore, known as a 'nanopore,' which functions as a biosensor and is embedded within a polymer membrane possessing electrical resistance (Wang et al., 2021).

Nanopores offer real-time detection meaning that the signals generated by the molecules passing through the nanopore are recorded and analysed immediately. This real-time analysis provides immediate feedback and insights into the molecular properties and dynamics (i.e. the interactions between the atoms of the molecule and with other molecules, over time), allowing for rapid decision-making and experimentation (Van Meervelt et al., 2017). Last but not least, nanopore sensing devices, particularly those based on solid-state nanopores, can be designed to be portable. This portability in combination with the real-time results, allows for on-site or field applications in various locations and environments (Chen et al., 2023).

Nanopores are versatile and can be used to sense a wide range of biomolecules, including DNA, RNA, proteins, small molecules, and even nanoparticles to better understand the living world. This broad applicability opens opportunities for various fields, including genetic studies, studying proteins, drug discovery, and diagnostics (Kobeissy et al., 2014).

Exemplifying the Versatility of Nanopores

Nanopore sensing is a versatile technology with wide-ranging applications in areas such as medicine, environmental monitoring, and research.

Nanopore sensing, process explained above, has brought a revolutionary change to DNA sequencing in the medical field. It can quickly and accurately read DNA code, which has opened up new possibilities for diagnosing diseases and personalised medicine. DNA code is unique to each individual, requiring treatments to be unique and specifically tailored also, especially in the cases of certain cancers and autoimmune diseases. Portable nanopore sequencers (i.e. pocket-size devices that read DNA code using nanopore technology) have made it possible to analyse DNA on the spot, giving real-time information about genetic variations and changes that occur in case of disease. Nanopore sensing is also useful for studying proteins, which are important biomolecules in our bodies. By modifying the surface of the nanopore, scientists can identify and study specific proteins and learn more about their structures and functions. This helps in finding markers for diseases and discovering new drugs. Another application of nanopore sensing is in observing how drugs are delivered into our bodies. Scientists can pass tiny particles or drug molecules through the nanopore and see how they interact and release over time. This helps in optimizing drug formulations and ensuring that they are effective. (Lastra et al., 2021).

Figure 4 - Four areas of research in which nanopores have great potential to contribute to new knowledge and new technologies beyond sequencing (Ying et al., 2022)

In the realm of environmental monitoring, nanopore sensing offers powerful tools for water quality analysis. Through the analysis of changes in ionic currents as water flows through a nanopore, researchers can detect and identify in real-time microorganisms (Acharya et al., 2020) that might be dangerous if present in the water. Air pollution monitoring is another vital application of nanopore sensing in environmental science. Nanopore sensors can detect and analyse airborne particulate matter such as pollen, smoke and dust (Tsutsui et al., 2019), all indicators of air quality and pollution.

Conclusion

In conclusion, nanopore sensing has revolutionised the field of biomolecule sensing by offering label-free, single-molecule detection and analysis. It enables the analysis of individual molecules in their natural state, provides real-time detection and immediate feedback, and offers versatility in sensing a wide range of molecules, which is crucial to understanding the wider picture of living organisms. Their ability to provide rapid and portable DNA/RNA sequencing, facilitate protein analysis, monitor drug delivery processes, analyse water, and air quality, enable single-molecule studies, and characterise nanoparticles has opened up new avenues for scientific discovery, healthcare diagnostics, and environmental sustainability. With ongoing advancements and increasing portability, nanopore sensing continues to pave the way for exciting discoveries and applications in the future.

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