Real Time Peak Detection for Translocation, Control, and Analysis in Solid State Nanopores
Engineering
EE 195, Senior Thesis
A nanopore is a small hole in a membrane, usually ranging from 1 to 100 nm in diameter. Most nanopores are created in synthetic or biological membranes that separate two volumes of electrolytic fluids. In such a setup, applying voltage across the membrane results in measurable ionic current flow through the pore. The amount of current observed depends on the conductivity of the electrolytic solution and the diameter of the pore, and remains relatively stable as long as the pore remains unobstructed. However, applying voltage across the pore can force charged particles to move through the pore, which disrupts the stable current with positive or negative current spikes. This property allows nanopores to detect and analyze nanoscale objects by monitoring the current through the pore. In particular, larger diameter nanopores (>10 nm diameter) can detect the passage of a large molecule like a DNA strand through the pore, while smaller diameter pores allow some physical properties of similarly sized molecules to be measured directly. These properties make nanopores of all sizes promising candidates for a variety of applications, including rapid, low cost DNA sequencing, label-free single molecule detection, and electrically controlled gating devices. In the Applied Optics group, nanopores are integrated with optofluidic devices to create a platform capable of analyzing and manipulating single molecules with both optical and pore based methods simultaneously.
Two ongoing projects in the Applied Optics group use this platform to investigate the integration of optics and nanopores. The first project aims to use an optical trap to enhance the translocation rate of DNA through the nanopore. This is done by attaching DNA strands to microbeads, confining the beads beneath a nanopore using the optical trap, and then using heat to release the DNA from the beads. This forms a volume of extremely high DNA concentrations around the nanopore, resulting in higher translocation rates. The second project aims to deliver individual molecules to a space of interest by allowing molecules to enter the space through a nanopore. Meanwhile, a microcontroller reading the current through the pore detects the current pulses that mark translocations, and shuts off the voltage across the pore when the desired number of translocations is detected. In the experiment, the space of interest was chosen to be a liquid core waveguide, which allowed the translocated particles to be detected by fluorescence spectroscopy.
The translocation rate enhancement project needed a system that could partially automate the process of measuring the thousands of translocation signals that were recorded in experiments. At the same time, the controlled delivery project needed a system that could detect translocation signals in real time and switch of the voltage across the pore at the desired number of translocations. Thus, the goal of this thesis was to aid in the development of these systems by creating a peak detection algorithm capable of accurately detecting translocation signals and applying this algorithm to both a real time detection system and post-experiment data analysis software.
To accomplish these goals, a peak detection algorithm was created and tested against simulated signals in Matlab. Then, the real time detection system was created by applying this algorithm to a detection program on a PSoC 5lp microcontroller, which was paired with a relay circuit capable of controlling the voltage applied across a nanopore This microcontroller system successfully detected translocations and gated the pore voltage, resulting in delivery of 1, 2, 3, and 4 DNA molecules to a liquid core waveguide. Further tests were conducted with the translocations of fluorescently labeled microbeads and lambda DNA molecules, and used fluorescence spectroscopy to confirm the controlled delivery of single particles through the pore. In addition the detection algorithm was applied to a Matlab script that shortened the analysis process for translocation containing signals from a 30 second per peak process to a 0.67 second per peak process, allowing analysis to proceed 45 times faster than previously. This program was subsequently used to analyze all data created by the translocation rate enhancement project.
