A Synthetic Electronic Nanopore for DNA
Sequencing and Stochastic Sensing
Mr. Aaron Choi, Computer Science, Sophomore
Mr. Davis Sneider, Biomedical Engineering, Sophomore
Mr. Saifuddin Aijaz, Chemical Engineering, Pre-Junior
Mentors:
Dr. David Wendell, Assistant Professor, Environmental Engineering
Dr. Vasile Nistor, Assistant Professor, Biomedical Engineering
Ms. Elizabeth Wurtzler, Graduate Student, Environmental Engineering
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Introduction
• Background
• Goals & Tasks
• Time Schedule– What we’ve done, where we’re going
• Inserting DNA– What we’re looking for, what we’ve found
• Findings
• Conclusion
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Current problem
• DNA sequencing can cost up to 10,000 dollars and take about a week
• Nanopore technology can save a lot of money and reduce the time to one day
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Nanopores: What are they?
• They are extremely small holes.
• They have potential applications for many kinds of developing technology
Oxford Nanopore Technologies
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Hydraphile Nanopore
• A synthetic nanopore, created by Dr. George Gokel at University of Missouri, St. Louis
• Lariat Ethers– Excellent cation selectivity– Excellent binding and release kinetics
Royal Society of Chemistry http://pubs.rsc.org/en/content/articlehtml/2000/cc/a903825f
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Size Comparison• The nanopore is said to be
approximately 8 picometers
• DNA has been shown to go through the nanopore and single stranded DNA is 1 nm
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Applications
• We could detect cancer earlier and much more efficiently
• DNA sequencing allows us to find many genetic disorders
• Ability to detect viruses
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Our Goals
• To determine which buffer works best
• To use the hydraphile nanopore for– DNA sequencing– Norovirus sensing
• Help to define the width of the hydraphile nanopore.
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Tasks• Use QuB to analyze data from four buffers
• Run items through nanopore– Ion Solutions– DNA – Norovirus
• Use passages to get an idea of how wide the nanopore is
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Time Schedule
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Conclusion From Buffers Tests
• Out of the four solutions used, it was determined that KCl is the best choice to use for nanopore sequencing as it gives a more stable membrane.
apcg.space.noa.gr
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Potassium Buffer
• 1M KCl Buffer, with 5mM Hepes
• Able to get data with ease
• Analyzing Data– Clampex
• 100< data points• Standard deviation
1.76 nanosiemens Glogster.com
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Painting the Membrane
• Take in and remove lipid hexane solution
• Create air bubble with pipet
• Wipe air bubble over membrane
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Inserting Nanopores
• Once a thin membrane is present, we then insert the hydraphile nanopore
• If membrane is too thick, nanopores won’t span length of membrane
Wikipedia http://en.wikipedia.org/wiki/Synthetic_ion_channels
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Nanopore Insertion
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Inserting DNA
• Dilute mixture– 2µL of DNA– 18µL of water
• Intake .5µL of mixture overtop of hole
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Detecting DNA Current Change
• Inserting DNA causes resistances in the current across the membrane– Negative charge across membrane
www.ks.uiuc.edu
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DNA Passing
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Resistance Zoomed19
What We Measured
• 2 major measurements– Blockage %– Dwell Time (ms)
• DNA length– 250 BP– 500 BP– 1,000 BP– 2,500 BP
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What Does It Mean & What Is It Useful For?
• Blockage %– Tells us how much of the nanopore has been blocked
– Helps us identify approximate width of DNA/RNA strand
• Event Duration– Tells us how long it took the DNA
segment to pass through the nanopore
– Helps us identify approximate length of the DNA/RNA strand
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References
• Gokel, George. Hydraphiles: Design, Synthesis and Analysis of a Family of Synthetic, Cation-conducting Channels. Tech. Royal Society of Chemistry, 24 Dec. 1999. Web. 13 June 2014.
• "Towards the 15-minute Genome." The Economist. The Economist Newspaper, 12 Mar. 2011. Web. 17 June 2014.
• Uddin A, Yemenicioglu S, Chen C-H, Corigliano E, Milaninia K and Theogarajan L. Integration of solid-state nanopores in a 0.5Â um CMOS foundry process. Nanotechnology. IOPScience, 31 October 2013. Web. 2 July 2014.
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Thank You!
• We would like to thank NSF for funding our research [Grant ID No.: DUE – 0756921]
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Questions?
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