Current blockade
A current blockade is the fall in ionic current through a pore while a molecule or particle occupies it. Its depth, usually written as a fraction of the open-pore current, its duration, and the shape of the trace between the fall and the recovery are the quantities measured in nanopore sensing, and in the Coulter counter before it. Under some conditions the current rises instead of falling, and the event is then a conductive pulse; Molecular Reality Corporation prefers the term "ionic modulation" for that reason.[1]
Origin of the signal
A voltage V across a pore of conductance G drives an open-pore current I0 = GV. One alpha-hemolysin channel in 1 M potassium chloride carried 120 pA at 120 mV, a conductance of 1 nS.[2] A molecule in the channel displaces conducting electrolyte, and the pore's resistance rises by an amount that, for a particle much smaller than the pore, is proportional to the volume of electrolyte displaced. This is the Coulter principle, and Maxwell's treatment of a suspension of insulating spheres gives it a shape factor of 3/2 for a sphere.[3] A long thin molecule lying along the current has a shape factor near 1, so the first estimate of its blockade is the ratio of its cross-section to that of the pore:
For double-stranded DNA 2.2 nm wide in a pore 5 nm wide this gives 19%. The pore's access resistance, which the molecule does not change, lowers the figure to about 17%, and a 10 nm pore gives 4.8% from area alone and 3.5% with access resistance included.[a] In absolute terms the drop in conductance, ΔG, falls from about 1.6 nS in a 4 nm pore to about 1.0 nS in a 10 nm pore in this calculation. Measurements on pores of 5 nm to 100 nm found the same trend and showed that access resistance has to be included to describe the conductance of the larger ones, contrary to earlier models that forecast a constant ΔG.[4]
Counted in charge, a 0.6 nA deficit lasting 10 µs is about 37,000 elementary charges that did not cross the pore.
Quantities
Software cuts a trace into events by event detection, and each event yields four numbers. Its depth, ΔI = I0 − Ib, is the gap between the open-pore current and the current Ib during the event, in picoamperes or nanoamperes; it is quoted as the fraction ΔI/I0 or, in the biological-pore literature, as the residual current Ib/I0. How long the event lasts is its dwell time, taken as the width at half the depth. Integrating ΔI over time gives the charge deficit. A fourth number, the interval to the next event, gives the rate of events. In stochastic sensing, where each event is a binding of the analyte to the pore, the rate reveals the concentration while the depth and duration help to identify the analyte.[5] In 1996 the blockade rate let a pore follow an enzyme at work: when ribonuclease was added to poly(U), the rate rose as long strands were cut into more, shorter molecules, then fell to zero once the fragments were too short to resolve.[2] The Coulter literature calls the same events resistive pulses, the 1996 DNA paper called them channel blockades, and the company's term is ionic modulation.[2][1]
Molecular Reality's public critique of its own world-model plans lists five classical features, "depth, FWHM dwell, area, asymmetry, inter-event time", and says that for isolated, clean, well-separated pulses a gradient-boosted classifier fed those five numbers is "the baseline and frequently the winner".[6] The company's guide describes the same trace in three acts: the current drops as the molecule enters, holds a plateau while it threads through, and springs back as it exits. It tells viewers of the live stream that "each dip is a candidate event, something passing through the sensing region".[7]
Fast events are attenuated by the bandwidth of the amplifier and filter, and look shallower than they are. The 1996 experiments used a low-pass Bessel filter set below three-eighths of a sampling rate of 64,000 samples a second, which is about 24 kHz, and the authors noted that the 100 µs collisions were barely twice the time resolution of their system.[2] The company's live trace is sampled at 200 kHz, one measurement every 5 µs.[7]
Levels
A single event can contain more than one level. In the first single-channel measurements on nucleic acids, the blockades of poly(U) RNA took the current down by 85% to 100% and lasted from several hundred to several thousand microseconds, with two example events of 300 µs and 1,300 µs for strands of about 210 nucleotides, and the lifetimes grew in proportion to polymer length and fell as the voltage rose.[2] The same paper separated three classes of event: brief collisions of about 100 µs that did not depend on length, translocations whose duration did, and occasional blockades of seconds that could be cleared by reducing the voltage to zero or reversing its polarity.[2] To show that the longer blockades were translocations, the experimenters counted by PCR the single-stranded molecules that reached the far chamber. With 35 channels in the membrane, about 1,300 blockades a minute corresponded to about 1,250 molecules a minute, within the ±30% precision of the blockade count, and double-stranded DNA produced only the brief collisions.[2]
Levels carry information about composition. An RNA strand of 70 cytosines followed by 30 adenines gave two current levels in each blockade, because the cytosine part forms a narrow single-stranded helix that can pass as it is and the adenine part forms a wider helix that must unwind first.[8] When an enzyme moves DNA through a pore a base at a time, the current steps from level to level, and in alpha-hemolysin about 12 bases contribute to each level and in MspA up to five.[8] A helicase that moves DNA through MspA in observable steps of about 0.33 nm produces a staircase of current levels, and the same arrangement read single amino acid substitutions in peptides.[9]
In solid-state pores a double-stranded DNA molecule can enter as a fold, putting two strands in the pore at once, and the event then shows a deeper level at about twice the single-strand blockade.[10] A knot in a long DNA molecule appears as a short spike on the trace, and the spikes can be counted.[11] A post of March 29, 2026 on Molecular Reality's member site reports the same signature at larger scale: "downward (resistive) spikes" as knots tied in a nylon filament were drawn through a macropore about 2 mm wide.[12] See Knot sequencing.
Proteins
A protein is not a uniform rod, and in a pore its blockade changes during the event as it tumbles in the electric field. A 2017 study used the zeptoliter sensing volume of a bilayer-coated solid-state pore to extract the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of single proteins from those modulations, all five from one set of events.[3] In ClyA, human and bovine thrombin, which are 86% identical in sequence, gave main current levels 26 pA apart at -50 mV.[13] In micron-scale apertures the shape of a pulse also depends on where the particle crosses, and particles that pass near the wall give pulses with two peaks.[14]
Conductive pulses
For DNA in solid-state pores the sign of the event depends on the salt concentration. Above about 0.4 M KCl the current falls and below it the current rises, and the two effects, the partial blocking of the pore and the counterions that screen the DNA backbone and carry current of their own, cancel at 370 ± 40 mM.[15] Counterions are the usual explanation of the rise. A 2022 study found that this theory "does not fit well with conductive events occurring via net neutral-charged protein translocation", and proposed instead that electro-osmotic flow and polarization effects in the pore produce conductive events.[16] The company's post "Clarifying why we say 'ionic modulation'" recommends that paper and gives its key point as "Analytes both block and enhance ionic current". The post adds that varying salt types and concentrations, with the asymmetry between the two sides of the pore, "will be an important dial for our Player Scientists to turn."[1]
Typical values
| Pore and analyte | Conditions | Signal | Source |
|---|---|---|---|
| A 76 µm hole in cellulose acetate film; red blood cells | Blood diluted several thousand times in 0.9% NaCl, October 1948 | Pulses lasting about 1 ms | [17] |
| Alpha-hemolysin; poly(U) RNA, mean length 210 nucleotides | 1 M KCl, 120 mV, 120 pA open-pore current | Current reduced by 85% to 100%; lifetimes 300 µs and 1,300 µs in the examples shown | [2] |
| ClyA; human and bovine thrombin, 86% identical in sequence | -50 mV | Main current levels 26 ± 1 pA apart | [13] |
| Solid-state pore; double-stranded DNA 16.5 µm long | KCl from 50 mM to 1 M | Current falls above 0.4 M and rises below | [15] |
| Pore diameter | Open-pore current | Area ratio | With access resistance |
|---|---|---|---|
| 4 nm | 1.1 nA | 30% | 27% |
| 5 nm | 1.7 nA | 19% | 17% |
| 10 nm | 5.9 nA | 4.8% | 3.5% |
See also
Notes
- ^ Calculated for a cylindrical pore 20 nm long in 1 M KCl at 10.5 S/m, with the conductance of the open pore taken as G = σ[4L/(πd²) + 1/d]−1 and the DNA as a coaxial cylinder that removes its cross-section from the channel term and leaves the access term unchanged. Counterion effects are ignored.[4]
References
- ^ a b c Kemmishtree (March 29, 2026). "Clarifying why we say "ionic modulation"". EQBio-OS.
- ^ a b c d e f g h Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. (1996). "Characterization of individual polynucleotide molecules using a membrane channel". Proceedings of the National Academy of Sciences 93 (24): 13770-13773. doi:10.1073/pnas.93.24.13770.
- ^ a b Yusko, E. C.; et al. (2017). "Real-time shape approximation and fingerprinting of single proteins using a nanopore (Supplementary Note 2)". Nature Nanotechnology 12 (4): 360-367. doi:10.1038/nnano.2016.267. PMID 27992411.
- ^ a b Kowalczyk, S. W.; Grosberg, A. Y.; Rabin, Y.; Dekker, C. (2011). "Modeling the conductance and DNA blockade of solid-state nanopores". Nanotechnology 22 (31): 315101. doi:10.1088/0957-4484/22/31/315101.
- ^ Gu, L.-Q.; et al. (1999). "Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter". Nature 398 (6729): 686-690. doi:10.1038/19491. PMID 10227291.
- ^ "Keltar's Critique v2: The Nanopore World Model (Hardened)". Molecular Reality Corporation. 2026.
- ^ a b "Keltar's Field Guide to Molecular Reality". Molecular Reality Corporation. August 2026.
- ^ a b Deamer, D.; Akeson, M.; Branton, D. (2016). "Three decades of nanopore sequencing". Nature Biotechnology 34 (5): 518-524. doi:10.1038/nbt.3423. PMID 27153285.
- ^ Brinkerhoff, H.; Kang, A. S. W.; Liu, J.; Aksimentiev, A.; Dekker, C. (2021). "Multiple rereads of single proteins at single-amino acid resolution using nanopores". Science 374 (6574): 1509-1513. doi:10.1126/science.abl4381. PMID 34735217.
- ^ Li, J.; Gershow, M.; Stein, D.; Brandin, E.; Golovchenko, J. A. (2003). "DNA molecules and configurations in a solid-state nanopore microscope". Nature Materials 2 (9): 611-615. doi:10.1038/nmat965. PMID 12942073.
- ^ Plesa, C.; et al. (2016). "Direct observation of DNA knots using a solid-state nanopore". Nature Nanotechnology 11 (12): 1093-1097. doi:10.1038/nnano.2016.153. PMID 27525473.
- ^ Kemmishtree (March 29, 2026). "Sequencing a string". EQBio-OS.
- ^ a b Soskine, M.; et al. (2012). "An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry". Nano Letters 12 (9): 4895-4900. doi:10.1021/nl3024438. PMID 22849517.
- ^ "Coulter Principle Short Course, Chapter 2". Beckman Coulter. Retrieved October 25, 2026.
- ^ a b Smeets, R. M. M.; et al. (2006). "Salt dependence of ion transport and DNA translocation through solid-state nanopores". Nano Letters 6 (1): 89-95. doi:10.1021/nl052107w.
- ^ Lastra, L. S.; et al. (2022). "On the origins of conductive pulse sensing inside a nanopore". Nature Communications 13: 2186. doi:10.1038/s41467-022-29758-8.
- ^ M. D. Graham (November 18, 2020). "The Coulter Principle: For the Good of Humankind, chapter 3, "Invention"". University of Kentucky. Retrieved October 25, 2026.
