A Synthetic Electronic Nanopore for DNA Sequencing and Stochastic Sensing Submitted to The 2014 Summer NSF REU Program Sponsored By The National Science Foundation Grant ID No.: DUE-0756921 College of Engineering and Applied Science University of Cincinnati Cincinnati, Ohio Prepared By: Mr. Aaron Choi, Chemical Engineering, University of Cincinnati Mr. Davis Sneider, Biomedical Engineering, The George Washington University Mr. Saifuddin Aijaz, Computer Science, University of Cincinnati Report Review By: Margaret J. Kupferle, PhD, PE REU Faculty Co-Mentor Department of Biomedial, Chemical, and Environmental Engineering University of Cincinnati July 3rd, 2014 Abstract _____________________________________________________________________________________ Nanopores are present in daily life, channels that control ion transport along with other substance concentrations, yet the importance and full practical use of these structures is just now being tapped. These ion channels carry currents across which can then be used to measure lengths of DNA and other molecules. This sequencing and sensing process is a science that is just being fully explored. At the University of Cincinnati researchers attempted to run certain saline solutions containing ions which would in turn allow the sensing and determination of which ions are present. In addition to ions, the used hydraphile nanopore used, developed by Dr. George Gokel, can be used to properly identify and norovirus. However, Dr. George Gokel’s nanopore is believed to be 6-8 picometers, but by passing DNA 1-2 nanometers wide through such pore, researchers hope to prove the pore width to be wider, and determine a better estimate. Introduction________________________________________________________________________________ Nanopores can shape the technology of the future. As DNA sequencing is costly and time consuming, we need to find a more efficient way to do it. Nanopores are the key to that problem. By threading DNA through a nanopore, we can detect what bases are which by looking at the changes the DNA makes in the ionic current. We can also use it to detect viruses and abnormalities that would be difficult to detect otherwise. The hydraphile nanopore is being tested as a synthetic nanopore to see how reliable and able it is to do such tasks. Starting June 9th, group three: Aaron Choi, Davis Sneider, and Saifuddin Aijaz, started their project and over the last two weeks until July 3rd, the group has made huge strides in the laboratory. Materials____________________________________________________________________________________ Requires 2.5µl, 10µl, and 1000µl pipettes for painting membranes and inserting nanopores, DNA, and ion solutions. Requires NaCl, KCl, and MgCl buffer solutions, inserted in the BCH1 chamber. Requires a dissecting scope as well as a Farraday cage. Data collection requires Digidata, Clampex, and Axopatch software and hardware. Methods_____________________________________________________________________________________ The basis of what the researchers did in weeks 3 and 4 revolved around painted a membrane. The fused lipid membrane with inserted nanopores allows the group to thread DNA through the nanopores. The reason why researchers want to thread DNA through nanopores is because DNA sequencing costs around $10,000 and takes up to eight days. Researchers want to be able to sequence DNA in a matter of hours even minutes and for less than $1,000. Nanopore sequencing is able to expedite the process of making DNA sequencing more efficient. By making DNA sequencing more efficient, we can detect cancer, hereditary illnesses and viruses. In weeks 3 and 4, the researchers moved on from learning and practicing painting membranes, and continued into data collection and analysis. The researchers first step in data analysis involved obtaining a constant data set and variable data sets. These sets were based on the buffer which was used : KCl, NaCl, or MgCl, and seawater. In order to obtain this information, the membranes were painted and once a thing membrane was present then the vesicles were entered. This gave a holding potential value for the nanopores entered and with this information the researchers were able to determine the amount of current jump which occurred from the opening of a single nanopore. Using Clampfit, the researchers took the average jump value for each buffer to determine which buffer was the most successful based on the most consistent value, and the ease of getting a sturdy membrane. Through this process there was also a mishap which occurred during midweek of week 3. Both electrodes attached silver wiring, used to conduct our current across the membrane, rendering the researchers incapable of conducting research. Two steps were taken in order to attempt and fix this issue, the first being the use of electrical type and wiring to reattach them. This proved to be unsuccessful, leading to the second solution. This solution involved soldering the broken wires together, which after multiple efforts proved to be successful. This allowed the current to pass across the membrane and the group was able to continue their studies. Results and Discussion___________________________________________________________________ Out of the four buffers which were tested, only three were successful, giving data showing that a current of ions was indeed able to flow through the hydraphile nanopore. The magnesium chloride buffer was unable to produce and data because the researchers believe that with the 2+ charge on the magnesium ion, it is too large to pass through the hydraphile nanopore. In comparison to K+ and Na+which have a single charge, the Magnesium ion’s two plus charge has more electrons in its valence electron cloud causing it to be larger, making it unable to pass through the nanopore and give a current. Out of the three buffers that were successful in allowing the ion current to pass through, there was a varying effect of the buffer on the current. After analyzing the data in Clampfit, the average jump for each nanopore opening was determined. The KCl buffer has an average resistance of 1.76 nano siemens and the NaCl buffer has an average resistance of 1.54 nano siemens. The buffer that is used for DNA sequencing will be determined by comparing their resistances. Whichever buffer has the least standard deviation will be used due to lower variance in measurements. The lower deviation allows the researchers have a more reliable buffer which in turn allows them to have more certainty in disturbances being blockages by DNA rather than instability in the membrane due to the buffer. This is vital while passage of DNA is the next step in the researcher’s project.