Nanopore

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Schematic of Nanopore Internal Machinery and corresponding current blockade during sequencing Nanopore-based single molecule mass spectrometry (5884864158).jpg
Schematic of Nanopore Internal Machinery and corresponding current blockade during sequencing

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.

Contents

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). [1]

It may be considered a Coulter counter for much smaller particles. [2]

Types

Organic

Inorganic

Nanopore based sequencing

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. [14] Nanopore sequencing can occur with bacterial nanopores as mentioned in the above section as well as with the Nanopore sequencing device(s) is created by Oxford Nanopore Technologies.

Monomer identification

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. [15] 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. [16] [17]

Ion current rectification (ICR) is an important phenomenon for nanopore. Ion current rectification can also be used as a drug sensor [18] [19] and be employed to investigate charge status in the polymer membrane. [20]

Applications to nanopore sequencing

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. [21] 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. [22] Understanding this also contributes more data to understanding the sequence of the peptide fully in its environment. [23] 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. [16] 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. [24]

Alternate applications

Since the discovery of track-etched technology in the late 1960s, filter membranes with needed diameter have found application potential in various fields including food safety, environmental pollution, biology, medicine, fuel cell, and chemistry. These track-etched membranes are typically made in polymer membrane through track-etching procedure, during which the polymer membrane is first irradiated by heavy ion beam to form tracks and then cylindrical pores or asymmetric pores are created along the track after wet etching.

As important as fabrication of the filter membranes with proper diameters, characterizations and measurements of these materials are of the same paramount. Until now, a few of methods have been developed, which can be classified into the following categories according to the physical mechanisms they exploited: imaging methods such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM); fluid transport such as bubble point and gas transport; fluid adsorptions such as nitrogen adsorption/desorption (BEH), mercury porosimetry, liquid-vapor equilibrium (BJH), gas-liquid equilibrium (permoporometry) and liquid-solid equilibrium (thermoporometry); electronic conductance; ultrasonic spectroscopy; and molecular transport.

More recently, the use of light transmission technique [25] as a method for nanopore size measurement has been proposed.

See also

Related Research Articles

<span class="mw-page-title-main">Nanopore sequencing</span> DNA / RNA sequencing technique

Nanopore sequencing is a third generation approach used in the sequencing of biopolymers — specifically, polynucleotides in the form of DNA or RNA.

<span class="mw-page-title-main">DNA sequencing</span> Process of determining the nucleic acid sequence

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

The Transporter Classification Database is an International Union of Biochemistry and Molecular Biology (IUBMB)-approved classification system for membrane transport proteins, including ion channels.

<span class="mw-page-title-main">Nanoporous materials</span>

Nanoporous materials consist of a regular organic or inorganic bulk phase in which a porous structure is present. Nanoporous materials exhibit pore diameters that are most appropriately quantified using units of nanometers. The diameter of pores in nanoporous materials is thus typically 100 nanometers or smaller. Pores may be open or closed, and pore connectivity and void fraction vary considerably, as with other porous materials. Open pores are pores that connect to the surface of the material whereas closed pores are pockets of void space within a bulk material. Open pores are useful for molecular separation techniques, adsorption, and catalysis studies. Closed pores are mainly used in thermal insulators and for structural applications.

Cytolysin refers to the substance secreted by microorganisms, plants or animals that is specifically toxic to individual cells, in many cases causing their dissolution through lysis. Cytolysins that have a specific action for certain cells are named accordingly. For instance, the cytolysins responsible for the destruction of red blood cells, thereby liberating hemoglobins, are named hemolysins, and so on. Cytolysins may be involved in immunity as well as in venoms.

<span class="mw-page-title-main">Pore-forming toxin</span> Protein-produced toxins that create pores in cell membrane

Pore-forming proteins are usually produced by bacteria, and include a number of protein exotoxins but may also be produced by other organisms such as apple snails that produce perivitellin-2 or earthworms, who produce lysenin. They are frequently cytotoxic, as they create unregulated pores in the membrane of targeted cells.

<span class="mw-page-title-main">Hemolysin</span> Molecule destroying the membrane of red blood cells

Hemolysins or haemolysins are lipids and proteins that cause lysis of red blood cells by disrupting the cell membrane. Although the lytic activity of some microbe-derived hemolysins on red blood cells may be of great importance for nutrient acquisition, many hemolysins produced by pathogens do not cause significant destruction of red blood cells during infection. However, hemolysins are often capable of lysing red blood cells in vitro.

<span class="mw-page-title-main">Nanofluidics</span> Dynamics of fluids confined in nanoscale structures

Nanofluidics is the study of the behavior, manipulation, and control of fluids that are confined to structures of nanometer characteristic dimensions. Fluids confined in these structures exhibit physical behaviors not observed in larger structures, such as those of micrometer dimensions and above, because the characteristic physical scaling lengths of the fluid, very closely coincide with the dimensions of the nanostructure itself.

<i>Staphylococcus aureus</i> alpha toxin

Alpha-toxin, also known as alpha-hemolysin (Hla), is the major cytotoxic agent released by bacterium Staphylococcus aureus and the first identified member of the pore forming beta-barrel toxin family. This toxin consists mostly of beta-sheets (68%) with only about 10% alpha-helices. The hly gene on the S. aureus chromosome encodes the 293 residue protein monomer, which forms heptameric units on the cellular membrane to form a complete beta-barrel pore. This structure allows the toxin to perform its major function, development of pores in the cellular membrane, eventually causing cell death.

'Staphylococcus aureus delta toxin is a toxin produced by Staphylococcus aureus. It has a wide spectrum of cytolytic activity.

Nanofluidic circuitry is a nanotechnology aiming for control of fluids in nanometer scale. Due to the effect of an electrical double layer within the fluid channel, the behavior of nanofluid is observed to be significantly different compared with its microfluidic counterparts. Its typical characteristic dimensions fall within the range of 1–100 nm. At least one dimension of the structure is in nanoscopic scale. Phenomena of fluids in nano-scale structure are discovered to be of different properties in electrochemistry and fluid dynamics.

John Hagan Pryce Bayley FRS, FLSW is a British scientist, who holds the position of Professor of Chemical Biology at the University of Oxford.

<span class="mw-page-title-main">Ion track</span>

Ion tracks are damage-trails created by swift heavy ions penetrating through solids, which may be sufficiently-contiguous for chemical etching in a variety of crystalline, glassy, and/or polymeric solids. They are associated with cylindrical damage-regions several nanometers in diameter and can be studied by Rutherford backscattering spectrometry (RBS), transmission electron microscopy (TEM), small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS) or gas permeation.

Oxford Nanopore Technologies plc is a UK-based company which develops and sells nanopore sequencing products for the direct, electronic analysis of single molecules.

David Wilson Deamer is an American biologist and Research Professor of Biomolecular Engineering at the University of California, Santa Cruz. Deamer has made significant contributions to the field of membrane biophysics. His work led to a novel method of DNA sequencing and a more complete understanding of the role of membranes in the origin of life.

Third-generation sequencing is a class of DNA sequencing methods currently under active development.

<span class="mw-page-title-main">Cynthia Burrows</span> American chemist

Cynthia J. Burrows is an American chemist, currently a distinguished professor in the department of chemistry at the University of Utah, where she is also the Thatcher Presidential Endowed Chair of Biological Chemistry. Burrows was the Senior Editor of the Journal of Organic Chemistry (2001-2013) and became Editor-in-Chief of Accounts of Chemical Research in 2014.,,

Jean-Pierre Leburton is the Gregory E. Stillman Professor of Electrical and Computer Engineering and professor of Physics at the University of Illinois at Urbana–Champaign. He is also a full-time faculty member in the Nanoelectronics and Nanomaterials group of the Beckman Institute for Advanced Science and Technology. He is known for his work on semiconductor theory and simulation, and on nanoscale quantum devices including quantum wires, quantum dots, and quantum wells. He studies and develops nanoscale materials with potential electronic and biological applications.

Lysenin is a pore-forming toxin (PFT) present in the coelomic fluid of the earthworm Eisenia fetida. Pore-forming toxins are a group of proteins that act as virulence factors of several pathogenic bacteria. Lysenin proteins are chiefly involved in the defense against cellular pathogens. Following the general mechanism of action of PFTs lysenin is segregated as a soluble monomer that binds specifically to a membrane receptor, sphingomyelin in the case of lysenin. After attaching to the membrane, the oligomerization begins, resulting in a nonamer on top of membrane, known as a prepore. After a conformational change, which could be triggered by a decrease of pH, the oligomer is inserted into the membrane in the so-called pore state.

Marija Drndic is the Fay R. and Eugene L. Langberg Professor of Physics at the University of Pennsylvania. She works on two-dimensional materials and novel spectroscopic techniques.

References

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  15. ↑ Li S, Cao C, Yang J, Long YT (2019-01-02). "Detection of Peptides with Different Charges and Lengths by Using the Aerolysin Nanopore". ChemElectroChem. 6 (1): 126–129. doi: 10.1002/celc.201800288 .
  16. 1 2 Wang Y, Gu LQ, Tian K (August 2018). "The aerolysin nanopore: from peptidomic to genomic applications". Nanoscale. 10 (29): 13857–13866. doi:10.1039/C8NR04255A. PMC   6157726 . PMID   29998253.
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  19. ↑ Guo Z, Wang J, Wang E (January 2012). "Selective discrimination of small hydrophobic biomolecules based on ion-current rectification in conically shaped nanochannel". Talanta. 89: 253–7. doi:10.1016/j.talanta.2011.12.022. PMID   22284488.
  20. ↑ Guo Z, Wang J, Ren J, Wang E (September 2011). "pH-reversed ionic current rectification displayed by conically shaped nanochannel without any modification". Nanoscale. 3 (9): 3767–73. Bibcode:2011Nanos...3.3767G. doi:10.1039/c1nr10434a. PMID   21826328. S2CID   205795031.
  21. ↑ Sutherland TC, Long YT, Stefureac RI, Bediako-Amoa I, Kraatz HB, Lee JS (July 2004). "Structure of Peptides Investigated by Nanopore Analysis". Nano Letters. 4 (7): 1273–1277. Bibcode:2004NanoL...4.1273S. doi:10.1021/nl049413e.
  22. ↑ Schmid, Sonja; Stömmer, Pierre; Dietz, Hendrik; Dekker, Cees (2021-03-09). "Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations". doi:10.1101/2021.03.09.434634.{{cite journal}}: Cite journal requires |journal= (help)
  23. ↑ Schiopu I, Iftemi S, Luchian T (2015-01-13). "Nanopore investigation of the stereoselective interactions between Cu(2+) and D,L-histidine amino acids engineered into an amyloidic fragment analogue". Langmuir. 31 (1): 387–96. doi:10.1021/la504243r. PMID   25479713.
  24. ↑ Li S, Cao C, Yang J, Long YT (2019). "Detection of Peptides with Different Charges and Lengths by Using the Aerolysin Nanopore". ChemElectroChem. 6 (1): 126–129. doi: 10.1002/celc.201800288 .
  25. ↑ Yang L, Zhai Q, Li G, Jiang H, Han L, Wang J, Wang E (December 2013). "A light transmission technique for pore size measurement in track-etched membranes". Chemical Communications. 49 (97): 11415–7. doi:10.1039/c3cc45841e. PMID   24169442. S2CID   205842947.

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