Many different nanopore research directions and applications beyond DNA sequencing

Many different nanopore research directions and applications beyond DNA sequencing

In a current one Nature nanotechnology In the study, researchers describe diverse applications of nanopore-based technology that go beyond deoxyribonucleic acid (DNA) sequencing. In particular, current research focuses on the advances of this technology in chemistry, biophysics and nanoscience.

Study: Nanopore-based technologies beyond DNA sequencing.Photo credit: Yurchanka Siarhei / Shutterstock.com

What are nanopores?

In a traditional application, the analytes of interest enter the nanopore under an applied current that alters the ion flow through the nanopore. This change in ion flux is reflected in a time-dependent current recording, which can be used to detect and characterize various biomolecules such as DNA, RNA, proteins, peptides, metabolites and protein-DNA complexes at the molecular level.

The type of nanopore used for a particular study depends on the analyte of interest, as both the nanopore and analyte dimensions should be comparable to produce a recordable change in ionic current.

For example, biological nanopores can detect biomolecules with diameters ranging from -1 to 10 nanometers (nm). In comparison, solid-state nanopores are used for optical applications including electron/ion millions, laser-based optical etching, and dielectric breakdown of ultrathin solid-state membranes.

Applications of nanopores

Although nanopores were originally developed to detect ions and small molecules, particularly for DNA sequencing purposes, the applications of this technology have expanded significantly.

The key advantages of nanopores that have contributed to their wide application include their ability to capture individual molecules sequentially and at high speed, convert both the structural and chemical properties of analytes into a measurable ionic current, and identify label-free species for signal amplification.

Structural analysis and sequencing of individual proteins

Solid-state nanopores can help extract the general properties of proteins such as volume, dipole and shape. Furthermore, ligands such as biotin, aptamers, protein domains or antibodies can bind directly 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 and provide information about protein activity, dynamics, and conformational changes. For example, by trapping a protein in a biological nanopore, researchers can obtain information about the protein's conformational changes and dynamics while it remains in the nanopore.

Although nanopores cannot provide information about the activities of individual enzymes, they can potentially monitor the formation of products following enzymatic reactions, especially when conventional spectroscopic assays are not available.

Single molecule chemistry

Biological nanopores designed to contain reactive sites are called protein nanoreactors. These specific nanopores could help analyze bond formation and bond breaking events of individual molecules attached to the inner wall of a nanopore while modulating the ionic current. Other applications of nanoreactors include the analysis of phytochemistry, stereochemical transformations, polymerization steps and a primary isotope effect.

Nanopores for studying biological processes

Cells have several nm sized pores in their membranes that serve as gates for molecular transport between cell compartments. To better understand the mechanisms involved in transporting biomolecules through these pores, they could be extracted from the cell and docked into planar lipid membranes. Unfortunately, this recovery approach is extremely difficult; Therefore, nanopores offer exciting opportunities for the study of cell biology.

Various nanopore-based engineered systems can mimic biological pores in vitro, such as asymmetric solid-state nanopores that could mimic switchable ion channels to study ion pumps and ion- and pH-controlled pores. Furthermore, synthetic DNA origami pores can also be used to mimic ligand-gated ion channels, while biological nanopores can be designed to mimic passive or active membrane transporters.

The nuclear pore complex (NPC), a larger pore that regulates the transport of proteins and RNAs between cellular compartments, can also be studied using biomimetic NPCs. Although extensive information about the biological function of NPCs is available, biomimetic NPCs can be used to better understand the specific transport properties of these biological pores.

Identification and quantification of biomarkers

Analyzing the presence of specific biomarkers in biomedical samples such as body fluids, tissue biopsies or other biological samples such as viruses, bacteria and cell cultures presents numerous challenges.

For example, target biomolecules in samples, many of which are nucleic acids or proteins, can be present at concentrations ranging from tens of attomolars (10–18 M) to the subnanomolar range (10–9 M). In addition, such clinical samples also contain various other biomolecules that can interfere with the nanopore sensor itself.

To overcome these limitations, various smart bioassays and devices have been developed that utilize nanopore sensing technology to analyze clinical samples. For example, novel microfluidic devices integrated with nanopore sensors can potentially be used for sample preparation or detection of analyte concentrations.

Furthermore, specific biochemical assays based on biological nanopores can improve molecular specificity while eliminating unwanted interactions with background molecules. This approach can also reduce the loss of target molecules during sample preparation while ensuring that the nanopore is protected from possible degradation by surrounding biomolecules.

Conclusions

Improvements in nanopore design will enable these technologies to advance and address scientific challenges. In addition, researchers expect that nanopores will find novel applications in a wide range of areas, from molecular sensing and sequencing to chemical catalysis and biophysical characterization.

Reference:

• Ying, Y., Hu, Z., Zhang, S., et al. (2022). Nanoporenbasierte Technologien über die DNA-Sequenzierung hinaus. Natur-Nanotechnologie. doi:10.1038/s41565-022-01193-2