Innovations in nanopore technology has advanced single-molecule studies for applications like sensor devices, and the further emergence of functionalized nanopores has increased the versatility of this technology. This article will provide an overview of functional nanopores and the challenges in the engineering these for advanced applications.
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Introduction to Functional Nanopores
Nanopore technology involving single-molecule detection originated from the recording technology of single-channel current, with the single-molecule analysis method being developed in 1966. Such a novel sensing detection technology offered advantages such as high sensitivity and versatility uncommon in industry at that time.
Nanopores can be broadly divided into two types including biological and solid-state nanopores, with biological nanopores also being referred to as transmembrane protein channels.
Contemporary nanopore technology has been developed further than the examination of biological ion channel function, with nanopore sensing being used for solid-state or biological nanopores with stable open channels.
An example of a ubiquitously used biological nanopore includes toxin proteins that are produced by pathogenic bacteria that have stable open channels, which provide low-noise ion currents when a voltage is applied across the membrane. These proteins have been repurposed for versatile applications including sensing, such as for single-molecule DNA sequencing.
Solid-State and Biological Nanopores
Solid-state nanopores are produced with a diameter of between 1 and 20 nm using materials such as silicon oxide or silicon nitride, however, due to effective single-molecule sensing requiring a slightly larger diameter than the analyte molecules, this scale may not be as valuable.
These nanopores comprise characteristics such as being more thermally and chemically stable than biological nanopores; however, with top-down approaches that are used to construct solid-state nanopores, such as with laser or electron beam etching, their structure at the atomic level cannot be controlled with precision. Overall, this means solid-state nanopores may not be well suited for synthetic modification.
Contrastively, biological nanopores, while less robust, consist of a structure that is more atomically precise due to being encoded by protein sequences, and so can be used diversely with chemical and genetic modifications.
Research into functionalized nanopores aimed to combine the advantages of both biological and synthetical channels to reduce limitations such as tunability and stability. This has led to the chemical and genetic modification of biological nanopores as well as introducing biomolecules to increase the functionality of solid-state pores.
Functionalized nanopores are created by incorporating biological and synthetic modifications into protein as well as solid-state nanopores, which allow functionalized nanopores to have increased functionality and efficacy. The alterations with biomolecules including DNA, peptides and antibodies that can provide solid-state nanopores, which are typically inert, with a capacity to have specific biomolecular and molecular interactions.
Additionally, biological nanopores can be altered with synthetic elements including crown ethers and cyclodextrins, which can allow for novel detection methods as well as real-time examination of single-level molecular interactions and chemical reactions. The combination of the two types of nanopores can increase robustness while also allowing for the nanopores to maintain their molecular recognition abilities.
Challenges of Functional Nanopores
While combining the two types of nanopores can seem simple in theory, the modification of existing nanopores is also associated with obstacles and limitations. There are many researchers that have advanced into this path, including the development of de novo synthetic protein nanopores.
Research involved in this area included the design and creation of a semi-synthetic self-assembled α-helical transmembrane protein that was based on the structure of an E. coli polysaccharide transporter. A 35-residue sequence was made using solid-state peptide synthesis and was observed to form discrete transmembrane channels that were expected to come together in an octamer in the lipid bilayer.
The channels were also observed to go through reversible conformational changes between being low-conductance and high-conductance, which demonstrated a lack in structural rigidity as well as reproducibility compared to a biological nanopore. As the work of this research progressed further, it was shown to hold potential for developing custom-designed protein channels.
Creating hybrid or semi-synthetic nanopores were also found to have challenges; in research that aims to combine biological and solid-state pores, these were found to be difficult to fabricate. Additionally, research that included separating active sections of the membrane of known proteins, these nanopores were found to have less reliable stability in comparison to native pores, as well as being difficult in predicting the structure. Research into combining existing proteins into a bespoke channel found similar challenges as native pores that have fast translocation that surpasses the limit of resolution.
The significance of nanopores is endless for a wide spectrum of applications, including the possibility to design synthetic protein pores from only synthetic materials with specific functions. This could prove to be useful for exploiting diverse chemical functionalities and characteristics of non-natural amino acids for advanced molecular biology uses, which have rarely been used to alter protein nanopores.
Additionally, the advancement in sensor technology with nanopores can also be used for the development of handheld devices that can provide fast and sensitive diagnostic information, improving healthcare of patients.
References and Further Reading
Branton, D., et al., (2008). The potential and challenges of nanopore sequencing. Nature Biotechnology, [online] 26(10), pp.1146–1153. doi.org/10.1038/nbt.1495
F. Cairns-Gibson, D. & Cockroft, S. L. (2022). Functionalised nanopores: chemical and biological modifications. Chemical Science, [online] 13(7), pp.1869–1882. doi.org/10.1039/d1sc05766a
Zeng, X., et al., (2021). Nanopore Technology for the Application of Protein Detection. Nanomaterials, 11(8), p.1942. doi.org/10.3390/nano11081942