In a recent Nature Nanotechnology study, researchers describe diverse applications of nanopore-based technology beyond deoxyribonucleic acid (DNA) sequencing. More specifically, the current research focuses on the advancements of this technology within chemistry, biophysics, and nanoscience.
Study: Nanopore-based technologies beyond DNA sequencing. Image Credit: Yurchanka Siarhei / Shutterstock.com
What are nanopores?
In a conventional application, analytes of interest will enter the nanopore under an applied current that changes the flow of ions through the nanopore. This change in ion flow is reflected as a time-dependent current recording that can be used to sense and characterize various biomolecules such as DNA, RNA, proteins, peptides, metabolites, and protein-DNA complexes at the molecular level.
The type of nanopore employed for a given study depends upon the analyte of interest, as both the nanopore and analyte dimensions should be comparable to produce a recordable change in ionic current.
Biological nanopores, for example, can recognize biomolecules with diameters within the range of -1 to 10 nanometers (nm). Comparatively, solid-state nanopores are used for optical applications, including electron/ion million, laser-based optical etching, and the dielectric breakdown of ultrathin solid membranes.
Applications of nanopores
Although nanopores were initially developed for sensing ions and small molecules, particularly for DNA sequencing purposes, the applications of this technology have expanded considerably.
Some of the key advantages of nanopores that have contributed to their widespread application include their ability to capture single molecules consecutively and at a rapid rate, convert both the structural and chemical properties of analytes into a measurable ionic current, and identify label-free species for signal amplification.
Single-protein structural analysis and sequencing
Solid-state nanopores can help extract the generic properties of proteins, such as volume, dipole, and shape. In addition, ligands, such as biotin, aptamers, protein domains, or antibodies, can directly attach to biological nanopores, even in the presence of complex media, such as serum.
In addition to identifying proteins, nanopores can act as single-molecule sensors to provide information on proteins' activity, dynamics, and conformational changes. By trapping a protein inside of a biological nanopore, for example, researchers can obtain information on the protein’s conformational changes and dynamics as it remains within the nanopore.
Although nanopores cannot provide information on the activities of individual enzymes, they may be able to monitor the formation of products following enzymatic reactions, mainly when conventional spectroscopic assays are unavailable.
Biological nanopores engineered to contain reactive sites are referred to as protein nanoreactors. These specific nanopores could assist in the analysis of bond-making and bond-breaking events of individual molecules attached to the interior wall of a nanopore as it modulates the ionic current. Additional applications of nanoreactors include the analysis of phytochemistry, stereochemical transformations, polymerization steps, and a primary isotope effect.
Nanopores to study biological processes
Cells feature several nm-sized pores within their membranes that act as gateways for molecular transport between cell compartments. To better understand the mechanisms involved in the transport of biomolecules through these pores, they could be extracted from the cell and docked within planar lipid membranes. Unfortunately, this reconstitution approach is extremely difficult; thus, nanopores offer exciting opportunities to study cell biology.
Various engineered nanopore-based systems can mimic biological pores in vitro, such as asymmetric solid-state nanopores, which could mimic switchable ion channels to study ion pumps and ion- and pH-gated pores. In addition, synthetic DNA origami pores can also be used to mimic ligand-gated ion channels, whereas biological nanopores can be designed to mimic passive or active membrane transporters.
The nuclear pore complex (NPC), a larger pore that regulates the transportation of proteins and RNAs between cellular compartments, may also be studied through biomimetic NPCs. Although considerable information is available on the biological function of NPCs, biomimetic NPCs can be used to better understand the specific transport properties of these biological pores.
Biomarker identification and quantification
Analyzing the presence of specific biomarkers within biomedical samples, such as bodily fluids, tissue biopsies, or other biological specimens, such as viruses, bacteria, and cell cultures, is associated with numerous challenges.
For example, target biomolecules within samples, many of which are nucleic acids or proteins, can be present in concentrations ranging from tens of attomolar (10−18 M) to the subnanomolar (10−9 M) range. In addition, such clinical samples also comprise various other biomolecules that may interfere with the nanopore sensor itself.
To overcome these limitations, various smart bioassays and devices utilizing nanopore sensing technology have been developed to analyze clinical samples. For example, novel microfluidic devices integrated with nanopore sensors can potentially be used for sample preparation or detecting analyte concentration levels.
Furthermore, specific biochemical assays based on biological nanopores can enhance molecular specificity while simultaneously eliminating unwanted interactions from background molecules. This approach can also reduce the loss of targeted molecules during sample preparation while ensuring that the nanopore is protected against any potential degradation from surrounding biomolecules.
With nanopore design improvements, these technologies will continue to evolve and address scientific challenges. Moreover, researchers anticipate that nanopores will find novel applications in a wide range of areas, from molecular sensing and sequencing to chemical catalysis and biophysical characterization.