1. October 26, 20151 NANOPORE PRESENTED BY: KAMYAR KHOSHNEVISAN DR.BORDBAR

2. 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. October 26, 20152

3. Classification:  Natural  Unnatural  Organic  Inorganic  Consist of carbon, silicon,silicates and polymers for aggregate material.  using zeolite in membrane October 26, 20153

4.  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) October 26, 20154

5.  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. October 26, 20155

6. -seven subunits -each subunit contributing with two β- strands to 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 October 26, 20156

7.  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) October 26, 20157

8. Basic properties of channels and pores. (a) Ion conduction; (b) ion selectivity; (c) gating;(d) channel block. October 26, 20158

9. 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. October 26, 20159

10.  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) October 26, 201510

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12.  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. October 26, 201512

13. I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. October 26, 201513

14. TThe prototype nanopore membrane chip contains four membranes, three of which have a 50 to 200 μm structured area. oEoEach 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. October 26, 201514

15. Nanopore membrane chip (pore arrays 10μm). October 26, 201515

16. Application in industry:  Purification with Nano Pores  NanoPore Thermal Insulation  Nanopore Technology for Biomedical Applications October 26, 201516

17. October 26, 2015 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. 17

18. October 26, 2015 A molecular model of translocation events 18

19. Translocation of single stranded DNA through a nanopore in Si3N4. Simulation conditions: Electrical field of 1.3x10 9 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. October 26, 201519

20. References : 1.C. R. Martin, P. Kohli, Nature Rev. DrugDisc. 2003, 2, 29–37. 2.G. E. Schulz, Biochim. Biophys. Acta 2002, 1565, 308–317. 3.K. S. Åkerfeldt, J. D. Lear, Z. R. Wasserman,L. A. Chung, W. F. DeGrado, Acc. Chem. Res. 1993, 26, 191–197. 4. H. Bayley, Curr. Opin. Biotechnol. 1999, 10,94–103. 5. S. Lee, T. Kiyota, T. Kunitake, E. Matsumoto,S. Yamashita, K. Anzai, G. Sugihara, Biochemistry 1997, 36, 3782–3791. 6.E. Matsumoto, T. Kiyota, S. Lee, G. Sugihara, S. Yamashita, H. Meno, Y. Aso, 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. October 26, 201520

21. 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 October 26, 201521

22. October 26, 2015 THANKS FOR YOUR PATIENCE 22