A nanopore is a pore of nanometer size. It may, for example, be created by a pore-forming protein or as a hole in synthetic materials such as silicon or graphene.

When a nanopore is present in an electrically insulating membrane, it can be used as a single-molecule detector. It can be a biological protein channel in a high electrical resistance lipid bilayer, a pore in a solid-state membrane or a hybrid of these – a protein channel set in a synthetic membrane. The detection principle is based on monitoring the ionic current passing through the nanopore as a voltage is applied across the membrane. When the nanopore is of molecular dimensions, passage of molecules (e.g., DNA) cause interruptions of the "open" current level, leading to a "translocation event" signal. The passage of RNA or single-stranded DNA molecules through the membrane-embedded alpha-hemolysin channel (1.5 nm diameter), for example, causes a ~90% blockage of the current (measured at 1 M KCl solution).

It may be considered a Coulter counter for much smaller particles.

The observation that a passing strand of DNA containing different bases corresponds with shifts in current values has led to the development of nanopore sequencing. Nanopore sequencing can occur with bacterial nanopores as mentioned in the above section as well as with the Nanopore sequencing device(s) created by Oxford Nanopore Technologies.

From a fundamental standpoint, nucleotides from DNA or RNA are identified based on shifts in current as the strand is entering the pore. The approach that Oxford Nanopore Technologies uses for nanopore DNA sequencing labeled DNA sample is loaded to the flow cell within the nanopore. The DNA fragment is guided to the nanopore and commences the unfolding of the helix. As the unwound helix moves through the nanopore, it is correlated with a change in the current value which is measured in thousand times per second. Nanopore analysis software can take this alternating current value for each base detected, and obtain the resulting DNA sequence. Similarly with the usage of biological nanopores, as a constant voltage is applied to the system, the alternating current can be observed. As DNA, RNA or peptides enter the pore, shifts in the current can be observed through this system that are characteristic of the monomer being identified.

Ion current rectification (ICR) is an important phenomenon for nanopore. Ion current rectification can also be used as a drug sensor and be employed to investigate charge status in the polymer membrane.

Apart from rapid DNA sequencing, other applications include separation of single stranded and double stranded DNA in solution, and the determination of length of polymers. At this stage, nanopores are making contributions to the understanding of polymer biophysics, single-molecule analysis of DNA-protein interactions, as well as peptide sequencing. When it comes to peptide sequencing bacterial nanopores like hemolysin, can be applied to both RNA, DNA and most recently protein sequencing. Such as when applied in a study in which peptides with the same Glycine-Proline-Proline repeat were synthesized, and then put through nanopore analysis, an accurate sequence was able to be attained. This can also be used to identify differences in stereochemistry of peptides based on intermolecular ionic interactions. Some configuration changes of protein could also be observed from the translocation curve. Understanding this also contributes more data to understanding the sequence of the peptide fully in its environment. Usage of another bacterial derived nanopore, an aerolysin nanopore, has shown ability having shown similar ability in distinguishing residues within a peptide has also shown the ability to identify toxins present even in proclaimed "very pure" protein samples, while demonstrating stability over varying pH values. A limitation to the usage of bacterial nanopores would be that peptides as short as six residues were accurately detected, but with larger more negatively charged peptides resulted in more background signal that is not representative of the molecule.

Nanopore are not only used for the sequencing of DNA, they are also used for the detection of many different biological entities from small molecules to large protein assemblies. Historically, alpha hemolysin was the first pore to demonstrate the potential of detecting the translocation (passage through the pore) of DNA and RNA strands. Nowadays, a whole array of both inorganic and biological nanopore are used for the detection of small molecules, peptides, proteins, biopolymers and sugars. These molecules are usually: