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NANOPORE

PRESENTED BY: KAMYAR KHOSHNEVISAN

DR.BORDBAR

What is a Nanopore? Nanopores occur in nature, and in the biological

literature they are simply known as pores when they are more than 1 nm or so in diameter, or channels when they are narrower. A nanopore is simply a small hole, of the order of 1

nanometer in internal diameter. Certain transmembrane cellular proteins act as nanopores, and nanopores have also been made by etching a

somewhat larger hole (several tens of nanometers) in a piece of silicon, and then gradually filling it in using ion-beam sculpting methods which results in a much

smaller diameter hole: the nanopore.April 20, 2023 2

Classification: Natural Unnatural Organic Inorganic

Consist of carbon, silicon,silicates and polymers for aggregate material.

using zeolite in membrane

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the natural channels that have been engineered as nanopores, the most prominent have been porins, proteins that control the permeability of the bacterial outer membrane,and a–hemolysin (aHL), a pore-forming toxin secreted by Staphylococcus aureus. Work on both these systems has been aided by high-resolution crystal structures.(figure)

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Protein pores are engineered by using mutagenesis and targeted chemical modification.The primary interest lies in engineering the interior (lumen) of the pores.

Mutagenesis can be used to introduce any of the twenty natural amino acids, which provide a variety of side chains of differing size, shape, polarity, and reactivity.

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-seven subunits-each subunit contributing with two β-strandsto form a 14-stranded β-barrel protein pore-cis entrance is ~70 Åabove the bilayer-total of ~100 Å-long lumen -average ~20 Åin internal diameter-the trans entrance is close to the bilayer surface

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What Can a Nanopore Do?

Ion channels open, conduct ions selectively, and close” The nanopores we are considering also

conduct ions, and even thoughthe diameters of the pores are larger than the typical ion channel, they do

exhibit at least modest ion selectivity.

Natural pores, such as gap junctions,can gate (open

and close in response to a stimulus), but so far little attention has been given to this issue with engineered

nanopores.(figure in next page)

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Basic properties of channels and pores. (a) Ion conduction; (b) ion selectivity; (c) gating;(d) channel block.

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What are the Potential Applications of Nanopores? separations with various modified inorganic

nanopores.

The separations have been based on several

properties including molecular mass,charge, hydrophobicity, and even stereochemistry.

similar separations might be carried out with

engineered protein nanopores.

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Another area of application of protein nanopores is in cell permeabilization. Because of its importance, the

utility of reversible plasma membrane permeabilization in cell and tissue preservation has

received the most attention.

Example of this application: Loading mammalian cells

with trehalose by using aHL-H5 pores,which can be blocked completely by Zn(II) ions(figure next page)

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Here example of fabrication procedure for ultrathin nanopore membranes, combining the techniques of

hot embossing for replication of nanopores and photolithography for production of the thin

membranes.The embossing masters are fabricated by electron beam lithography allowing customisation of

the design of the nanopore arrays.

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In a first step, the origination of nanostructures is

carried out using electron beam lithography to produce an embossing stamp. Here the design can

be tailored to the customers requirements.

For nanopore replication, the stamp consists of nano- sized pillars,which are pressed into a polymer resist that has been softened by applying heat. The final steps of the fabrication process involve etch processes for patterntransfer and photolithography with micromachining to produce the final 260 nm thick membrane.

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The prototype nanopore membrane chip contains four

membranes, three of which have a 50 to 200 μm

structured area.

o Each membrane contains pores of a different diameter of 550, 330 and 140 nm, and centre-to-

centre distances of 0.3 to 1 μm, 0.5 to 1 μm and 1 to 2 μm, respectively. The fourth membrane was left

unstructured as a control.

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Nanopore membrane chip (pore arrays 10μm).

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Application in industry:

Purification with Nano Pores

NanoPore Thermal Insulation

Nanopore Technology for Biomedical Applications

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Schematic of gas permeation in a pressure gradient through a membrane represented by a cylindrical pore of radius H, length L. The hardcore diameter of the spherical gas molecules is σ.

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A molecular model of translocation events

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Translocation of single stranded DNA through a nanopore in Si3N4. Simulation conditions: Electrical field of 1.3x109 V/m generates a voltage bias of 21 V across the membrane; 1.3±0.1 nm diameter pore; 5.2 nm thick membrane; single stranded (dC)20, 1M solution of KCl; 40125 atoms simulated; total simulation time is 1.3 ns; NvT ensemble.

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References :

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2.G. E. Schulz, Biochim. Biophys. Acta 2002, 1565, 308–317.

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4. H. Bayley, Curr. Opin. Biotechnol. 1999, 10,94–103.

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Biochemistry 1997, 36, 3782–3791.

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H. Sakamoto, H. M. Ellerby, Biopolymers2001, 56, 96–108.

7.S. Cheley, O. Braha, X. Lu, S. Conlan,H. Bayley, Protein Sci. 1999, 8, 1257–1267.April 20, 2023 20

8. J.K. Percus, in: H.L. Frisch, J.L. Lebowitz (Eds.), The EquilibriumTheory of Classical Fluids, W.A. Benjamin, New York, 1964, pp.

9. Vercoutere, W. et al. (2001) Rapid discrimination among individual DNA hairpin molecules at single-necleotide resolution using an ionchannel. Nat. Biotechnol. 19, 248–252

10. Howorka, S. et al. (2001) Sequence-specific detection of individual DNA

strands using engineered nanopores. Nat. Biotechnol. 19, 636–639

11.Deamer, D.W. and Akeson, M. (2000) Nanopores and nucleic acids:

prospects for ultrarapid sequencing. Trends Biotechnol. 18, 147–151

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THANKS FOR YOUR PATIENCE

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