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Nanopore Based Characterisation of Viruses and Nanoparticles

Nanopore Based Characterisation of Viruses and Nanoparticles. Hans van der Voorn - hans@izon.com. Contents. The Science The Technology The Applications The Future Our Proposition. SIOS: Resistive pulse sensing using a tunable nanopore. Uses: Particle counting

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Nanopore Based Characterisation of Viruses and Nanoparticles

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  1. Nanopore BasedCharacterisation of Viruses and Nanoparticles Hans van der Voorn - hans@izon.com

  2. Contents • The Science • The Technology • The Applications • The Future • Our Proposition

  3. SIOS: Resistive pulse sensing using a tunable nanopore • Uses: • Particle counting • Particle characterisation (e.g. sizing) • Specific particle detection (e.g. virology) • Direct sequencing of DNA (proof of concept only) • Types of nanopores • Biological • e.g. Alpha Haemolysin, Ion Channels • Solid state pores e.g. • Silicon Nitride (FIB) • Carbon nanotubes • Tunable pores

  4. Resistive Pulse Sensing Please view the SIOS animation through our website by clicking on the following link: View SIOS Animation >>

  5. Particle Transport Mechanisms • Particle Flux (particles/area/time) = Electrokinetic + Convection • + Diffusion • The ability to vary electrophoretic force and pressure provides a powerful measurement platform for many types of nanoscale particles. • Measurable characteristics include: • concentration • size • polydispersity • electrophoretic mobility • morphology • All these are available on a particle by particle basis

  6. Customers & Research Partners University of Otago Industrial Research Ltd MacDiarmid Institute for Nanotechnology Cawthron Institute University of Auckland Victoria University University of Canterbury ESR (NCBID) NIWA Massey University National School of Pharmacy University of Queensland (AIBN) AIST (Japan) UC Santa Cruz Oxford University Chemistry (UK) BegbrokeNano (Oxford, UK) Oxford University DPAG A*Star Institutes, Singapore MIT NIOZ (Netherlands) Max Planck Institute INBT, Johns Hopkins University NIST ATCC (USA) Georgia Tech International New Zealand

  7. Contents • The Technology

  8. Tunable Nanopore Technology • Nanopore fabricated in flexible thermoplastic polyurethane • Tunable, real-time measurement • Single particle detection capability • Capable of measuring almost any type of nanoparticle • Wide range of electrolytes

  9. Conical Shaped Nanopore SEM imaging Confocal Microscopy imaging

  10. Detecting blockade events Fitted baseline calculated over a user defined analysis window Baseline ionic current Blockade Event

  11. Blockade event features Duration Magnitude

  12. Tunability - Particle Gating x = 46.6mm, i = 144nA x = 46.0mm, i = 116nA Applying stretch to nanopore allows the system to be tuned to detect or gate particles as required x = 45.3mm, i = 91nA x = 44.7mm, i = 74nA

  13. Contents • The Applications

  14. What samples can be measured? • Size range ~ 40nm – 6µm • Concentration down to ~ 1x105 particles/mL • Wide range of particles, synthetic and biological • Viruses e.g. Adenovirus, Lentivirus, VZV, Dengue • Bacteria (Prochlorococcus) • Phages • Polymer particles • Metallic nanoparticles, Magnetic particles, Silica • Liposomes, exosomes

  15. Real-time Reaction Monitoring The interaction of two particles can result in a measurable change in key parameters: frequency time of flight magnitude

  16. Real-time Reaction Monitoring This can be used to: confirm the occurrence of a binding interaction compare properties of the two samples detect the presence of molecules that may otherwise be below the detection limit of the instrument monitor the binding interaction event in real-time particle-by-particle by combining the reactants in the instrument

  17. + avidin Detection of Chemical Reaction • Interaction of Avidin (~ 3 x 4 x 6.5 nm) + carboxylated polystyrene nanoparticles (184 nm ± 8.5)  pre incubation Lower Avidin concentration (50ng/1µL) – reduction in net surface charge due to binding results in increased translocation duration. High Avidin concentration (500ng/1µL) – surface charge ‘neutralised’, frequency quenched

  18. In-situ reaction monitoring Real time interaction of avidin and polystyrene constituents in upper fluid cell: 10ng/1µL Avidin introduced Polystyrene only

  19. Observing aggregation effects

  20. ImmunoNanoMetrology Please view the INM animation through our website by clicking on the following link: View INM Animation >>

  21. Particle concentration measurement • Particle flux (for electrophoretic transport • Rapid concentration analysis of samples • Applicable over range of tunable parameters (voltage, stretch)

  22. C’ Freq’’.C’ C’’ = Freq’ Variable Pressure Method For Concentration Determination - Particle concentration independent of particle size, type, charge Plot of blockade freq vs applied pressure Unknown particle with unknown concentration Y = m’’x Calibration particles, PS 220 nm etc Blockade freq counts/ min Known concentration Y = m’x 0 Applied Pressure/ kPa

  23. Particle charge/mobility analysis Variable Pressure Method (VPM) Plot of Blockade frequency vs Applied Pressure Blockade freq counts/ min Pressure 0 P = 0, Electrophoresis Mode Pressure force required to oppose electrophoretic force

  24. Solution: Particle Surface Properties 1 – Variable Voltage Method E Plot of Blockade Width (Duration) vs Applied Voltage v Electrophoretic Mobility Blockade width (duration) At a particular fixed Pressure +1V -1V count Applied Voltage

  25. Size Distribution Analysis Blockade magnitude proportional to particle volume. Distinguish particle populations based on size

  26. Solution: Size Analysis Standard line Trimodal mixture

  27. 3D Topology Analysis 56nm PS 89nm PS

  28. Particle Morphology Spherical Particles Polyaniline polymer tubes

  29. Contents • The Future

  30. The Future Short Term: Software enhancements Automation/feedback control Medium/Long term: Inline process QA/monitoring Environmental testing Miniaturisation, PoC diagnostics

  31. IZON Packages • Responsive quotes • Hardware, Software, Technical Support, Training • qNano / qViro Instruments • Variable Pressure Module (VPM) • Izon nanopores • Supportive link with IZON team • Free software upgrades (automatic online)

  32. Academic Pricing (Now) • Instrument Package - US$16000 • Apertures - US$55 each • Support - Included • Softwares Upgrades - Included • Onsite Training - As reqd • Lease to buy option

  33. Summary SIOS will become the accepted method of concentration analysis Detailed description of particles Solution for complex particle systems Nanoparticle drug delivery Diagnostics Bioparticles Multi-dimensional analysis Surface functionalisation

  34. Contacts • www.izon.com • hans@izon.com • info@izon.com • US Office: C/- BridgePath Scientific, Maryland • UK/Europe office due August 2010

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