Glossary of nanopore terms

This glossary of nanopore terms defines the words used in sensing single molecules and particles by the ionic current through small holes, from the physics of the salt water to the electronics that read the current. A nanopore is a hole about 1 to 100 nm across in a thin membrane between two chambers of electrolyte; the Coulter principle, on which it works, was first applied to blood cells through apertures about 100 µm wide.

Unless stated otherwise, values are for 1 M potassium chloride at about 25 °C. Terms coined by Molecular Reality Corporation are marked as company terms; the rest of the company's vocabulary is in the glossary of Molecular Reality jargon.

Access resistance

The resistance of the electrolyte outside the mouths of a pore, where current spreading through the reservoir has to converge on the opening. For a circular pore of radius a it is 1/(4σa) on each side, where σ is the conductivity of the electrolyte, and it dominates in pores much wider than they are long.^[1]

Aerolysin

A pore-forming toxin of the bacterium Aeromonas hydrophila. Its long, narrow and strongly charged channel resolves short peptides and oligonucleotides.

Ag/AgCl electrode

Silver coated with silver chloride. It carries current into a chloride solution by converting silver to silver chloride at one electrode and back at the other, and so barely polarizes while both last.

Alpha-hemolysin

A toxin of Staphylococcus aureus whose seven subunits form a channel 1.4 nm wide at its narrowest point. In 1996 it became the first pore to register single strands of DNA and RNA.^[2]

Analog front end (AFE)

The electronics between the electrodes and the digitizer: the bias source, the current amplifier and the filters.

Analog-to-digital converter (ADC)

The chip that turns the amplified signal into numbers, at a fixed sampling rate and a resolution counted in bits.

Axopatch 200B

A cooled patch clamp amplifier sold by Molecular Devices, the amplifier of many academic nanopore studies.

Bandwidth

The range of frequencies a measurement passes. A wider bandwidth resolves shorter events and admits more noise; when the capacitance of the chip dominates, the noise current grows as the bandwidth to the power 3/2.

Basecalling

Translating a current trace into the sequence of bases of a DNA or RNA strand, now done with neural networks.

Bessel filter

A low-pass filter with nearly constant delay at all frequencies it passes, so that it rounds the edges of a square pulse without ringing. It is the usual filter in patch-clamp and nanopore amplifiers.

Biological nanopore

A protein channel, about 1 nm wide at its narrowest point, set in a lipid or polymer membrane.

Blockade

See current blockade.

Brownian motion

The random jostling of small particles by the molecules of the liquid. It brings molecules to the pore and makes each passage different.

Capture radius

The distance from the pore inside which its electric field, rather than diffusion, governs a molecule's motion. For DNA it is about d²μΔV/(8LD), for a pore of diameter d and length L, voltage ΔV, electrophoretic mobility μ and diffusion coefficient D, and can exceed a micrometer.^[3] See capture rate (nanopore).

Capture rate

The number of molecules entering a pore each second, which rises with concentration and voltage.

Cis and trans

The two chambers on either side of the membrane. The sample is usually added to the cis chamber and driven toward trans.

Clogging

Blocking of a pore by a molecule or particle that does not leave, often cleared by reversing or pulsing the voltage.

Conductive pulse

A rise in current while a molecule is in the pore, seen at low salt concentrations, when the counterions the molecule carries add more current than the electrolyte it displaces removes.

Controlled dielectric breakdown (CDB)

Making a pore by holding a voltage across a thin insulating membrane in salt water until it fails at a single point, a method published in 2014 by a group at the University of Ottawa.

Coulter counter

The instrument that counts and sizes blood cells by the dip in current as each crosses an aperture about 100 µm wide. Wallace H. Coulter's patent on it was granted on October 20, 1953.^[4]

Coulter principle

A particle in an aperture filled with electrolyte raises its resistance, by an amount set mainly by the particle's volume.

CsgG

A secretion channel of the bacterium Escherichia coli whose constriction is 0.9 nm wide. Oxford Nanopore Technologies adopted engineered versions for its sequencers in 2016.

Current blockade

The fall in ionic current while a molecule or particle occupies a pore, described by its depth, its duration and the shape of the trace between fall and recovery.

Debye length

The distance over which the ions of an electrolyte screen an electric field: about 0.3 nm in 1 M salt and about 3 nm in 10 mM.

Dielectric noise

Noise from energy lost in the capacitance of the membrane and its support, rising in proportion to frequency.

DNA barcode

A DNA carrier with structures, such as hairpins, at set positions along it, whose pattern of spikes in the current encodes bits. A 2016 study read three-bit barcodes and used them to detect several proteins at once.^[5]

DNA origami

DNA folded into designed shapes, used as carriers for analytes and as pores in its own right.

Dwell time

The time a molecule spends in the pore during one event, which is the width of its pulse.

Electric double layer

The cloud of counterions that gathers at a charged surface in an electrolyte, about one Debye length thick.

Electroosmotic flow

Liquid dragged along a charged channel wall by the moving ions of its double layer. It can carry a molecule with the electric force or against it.

Electrophoresis

The motion of a charged molecule in an electric field. DNA, negatively charged, moves toward the positive electrode.

Event

One visit of a molecule or particle to the pore, seen as a pulse in the current.

Event detection

Finding events in a trace, most often by a threshold set some number of standard deviations of the noise away from the baseline.

Excluded volume

The volume of electrolyte a particle displaces in the pore, which sets the height of its pulse.

Feedback resistor

The resistor that sets the gain of a transimpedance amplifier. A larger resistor makes less noise and allows less bandwidth: 1 GΩ contributes about 4 fA/√Hz of thermal noise, 1 MΩ about 128 fA/√Hz.

Flicker noise

Noise whose power rises as frequency falls, as 1/f, the dominant noise of most pores at low frequencies.

Flossing

Moving one molecule back and forth through a pore, or between two pores, so that it can be read many times.

Flow cell

The holder that clamps the membrane chip between two reservoirs of electrolyte and carries the electrodes.

Focused ion beam (FIB)

A beam of ions, usually gallium, that mills holes down to tens of nanometers wide in thin membranes.

Folded passage

A DNA molecule that enters a pore by its middle instead of an end, giving a step to about twice the usual blockade depth.

Four-electrode cell

A cell with separate electrodes for passing current and for sensing voltage, which keeps the polarization of the current-carrying electrodes out of the measured voltage.

Fractional blockade

The depth of a blockade as a fraction of the open-pore current. To first order, double-stranded DNA 2.2 nm wide blocks (2.2/5)², or 19 percent, of a pore 5 nm wide; measured values are smaller.

Gating

Spontaneous switching of an empty pore between levels of conductance, a nuisance in some protein pores and in pores with hydrophobic walls.

Glass nanopipette

A glass capillary heated and pulled to a tip with an opening a few tens of nanometers wide or less, a solid-state pore that needs no clean room.

Graphene nanopore

A pore in a sheet of graphene a single atom, about 0.34 nm, thick.^[6]

Ion channel

A protein pore in a cell membrane that passes ions, the natural model for every nanopore.

Ion current rectification

Unequal currents at voltages of equal size and opposite sign, seen in conical or strongly charged pores.

Ionic current

Current carried by ions moving through a solution. In potassium chloride the two ions carry nearly equal shares.

Johnson-Nyquist noise

The thermal noise of any resistance, with a current spectral density of √(4kT/R).

Juxtapore

Company term: a pore formed between the edges of two solid surfaces sliding alongside each other.

k-mer

The few bases that sit in the narrowest part of a protein pore at once and together set the current at each step of a sequencing read.

Label

A chemical tag attached to particular bases or residues so that a pore can tell them apart, such as osmium tetroxide bipyridine on DNA.

Lambda phage DNA

The genome of a bacterial virus, 48,502 base pairs and about 16 µm long, a standard test molecule for solid-state pores.

Lipid bilayer

A membrane two molecules thick, about 5 nm, into which protein pores insert themselves.

Manning condensation

The binding of counterions along a highly charged polymer, which leaves DNA with about a quarter of its bare charge of two electrons per base pair.

Mechanical nanopore

A pore whose opening can be changed after fabrication by moving, stretching or bending the material around it.

Microfluidics

Channels tens to hundreds of micrometers wide that bring samples to the pore and wash them away.

Molybdenum disulfide nanopore

A pore in a sheet of MoS₂ three atoms thick. Such pores distinguished single nucleotides in 2015.^[7]

Motor protein

An enzyme, usually a helicase or polymerase, that ratchets a strand through a protein pore about one base at a time, slow enough to read.

MspA

A porin of Mycobacterium smegmatis whose constriction is 1.2 nm wide and no more than 0.6 nm long.

Nanobubble

A bubble of gas nanometers across that can lodge in a pore and cut its current. Its internal pressure is high: about 28 MPa, some 280 atmospheres, for a bubble 5 nm in radius.^[8]

Nanofluidics

The behavior of liquids and ions in channels a few nanometers across, where surface charge and the double layer govern transport.

Nanopore fabrication

The making of single pores, by drilling with electron or ion beams, etching, dielectric breakdown or pulling glass.

Nanopore noise

The random fluctuation of the current through a pore, the sum of flicker, thermal, shot, dielectric and capacitive noise.

Nanopore sequencing

Reading the bases of DNA or RNA by passing single strands through a pore about a nanometer wide and recording the current.

Open-pore current

The steady current through an empty pore, against which blockades are measured.

Patch clamp

The technique, and the class of amplifier, for holding a voltage across a membrane and measuring the picoampere currents through it, devised to record ion channels and borrowed by nanopore research.

Persistence length

The length over which a polymer stays roughly straight; for double-stranded DNA about 50 nm.

Picoampere

10⁻¹² ampere, about six million elementary charges per second.

Ping-pong sensing

Measuring one particle or molecule repeatedly by reversing the voltage after each pass.

Polystyrene bead

A sphere of known size, from tens of nanometers to micrometers across, used to calibrate pores.

Pore conductance

The current per volt through an open pore. For a cylinder of diameter d and length L it is σ[4L/(πd²) + 1/d]⁻¹, with the second term from access resistance. A pore 5 nm wide and 10 nm long conducts about 15.5 nS, or 3.1 nA at 200 mV, which is about 19 billion ions a second.^[9]

Potassium chloride

The usual electrolyte, chosen because its two ions move at nearly the same speed. At 1 M and 25 °C its conductivity is about 11 S/m.

Protein sequencing

Reading the order of amino acids in a protein, which nanopores have approached but not achieved for whole proteins.

QuipuNet

A convolutional neural network, published in 2018, that classifies the current signals of DNA carriers with three-bit barcodes; also the name of Molecular Reality Corporation's viewer of the same public data.^[10]

Recapture

Pulling a molecule back into the pore after it has passed, by reversing the voltage within milliseconds.^[11]

Resistive pulse sensing

Counting and sizing particles by the brief change in current as each crosses a small aperture.

Sampling rate

The number of current measurements per second. A pulse must span several samples to be measured, so a 100 kHz digitizer, with samples 10 µs apart, cannot size a pulse of a few microseconds.

Shot noise

Noise from the discreteness of the charge carriers, with a current spectral density of √(2qI): about 23 fA/√Hz at 1.7 nA.

Signal-to-noise ratio

The depth of a pulse divided by the noise of the baseline at the bandwidth used.

Silicon nitride membrane

A film of silicon nitride, typically 10 to 50 nm thick, suspended over a window in a silicon chip; the usual substrate for solid-state pores.

Sliding occlusion

Company term: adjusting a pore by sliding one perforated membrane across another.

Solid-state nanopore

A hole from about a nanometer to a few hundred nanometers wide in a synthetic membrane or the tip of pulled glass.

Surface charge

The charge of the pore walls, which sets the double layer and the electroosmotic flow. Silicon nitride is negatively charged at neutral pH.

Transimpedance amplifier (TIA)

An amplifier that converts a current into a voltage, with a gain in ohms set by its feedback resistor.

Translocation

The passage of a molecule from one side of the membrane to the other through the pore. DNA crosses a bare solid-state pore at about one to tens of bases per microsecond.

Tunable resistive pulse sensing

Resistive pulse sensing through an elastic pore that is stretched to change its size.

Voltage clamp

Holding the voltage across a membrane constant while measuring the current; the job of a patch-clamp amplifier.

Weep moat

Company term from the design of the MR1 Nanopore Reader: a sensing trace around the wet zone of a cartridge that detects leaking salt water as a fall in resistance, designed to read more than 10 GΩ dry and under 1 kΩ wet.^[12]

Wetting

Filling a pore with electrolyte. Small and hydrophobic pores resist it, and a pore that has not wetted carries little or no current.

Zeptoliter

10⁻²¹ liter, the volume of a cube 10 nm on a side. In 2017 two pores were joined by a bridge of liquid of about this volume, which trapped single DNA molecules between them.^[13]

• History of nanopore sequencing
• List of holes
• Machine learning on nanopore signals