PERSONAL INSPIRATION BEHIND MY PROJECT
Over the past few years, my Mayo Clinic mentor, Dr. Chris Pierret, taught me about genetics and PCR, and the problem of disease outbreaks in developing nations. He is someone whom I admire as both a scientist and friend. He has traveled to India and Africa, and shared stories about diseases killing thousands due to the lack of affordable technology. He told me that these experiences were life changing to him and how he wanted to focus his scientific efforts on helping these people in developing regions of the world. Hearing about his experiences, and specifically about a young boy who perished, motivated me to want to use my science, technology, engineering and math skills to develop technology that could potentially help these people.
When people in an area get sick with a viruse like Dengue Fever, which is spread by mosquitoes, one of the most important steps is to identify whether it really is Dengue. If you can identify it quickly, you can do more to stop it from spreading. Fast disease identification is the key to stop it from spreading.
There are a couple ways to identify diseases. Some methods are very slow and require growing the virus, which may take a week (or two weeks in remote areas). Genetic identification is currently the fastest method-- we can verify a virus is present in a few hours. If someone in New Delhi, India gets very sick, hospitals and public officials need to know immediately whether it's Dengue, so they can go where the person lives and eliminate the mosquitos that carry it.
Genetic identification also gives more information than other tests. Dengue Fever comes in 4 basic serotypes or flavors and some serotypes are more dangerous to some populations. So genetic identification is faster and more specific.
WHAT DOES A THERMOCYCLER DO?
PCR is a genetic technique that's a vital part of identifying diseases. You can start with the DNA from an infected person (e.g. a swab of saliva), but you don't yet have enough of the right kinds of DNA or RNA to perform a test. To detect whether the Dengue virus is present, you first need to make billions of copies of precise sections of the virus DNA or RNA used to identify the virus. For that, we use PCR-- a super DNA copy machine.
A thermocycler is the key instrument used in PCR. It cycles a mixture of DNA and other chemicals (about 1/2 drop in test tubes) between 3 temperatures (e.g. 55 C, 75 C, 95 C) to perform the copy process. Every time you cycle through all 3 temperatures, you DOUBLE the amount of DNA!! About 35-40 full temperature cycles can take 1.5 - 2 hours, and it must not be interrupted-- a challenge in areas where electrical power is unreliable.
So a thermocycler is the instrument that makes PCR happen and PCR is the vital step in isolating and making copies of exact fragments of DNA needed to identify a disease. A technique called RT-PCR has been perfected to rapidly identify Dengue fever. Other techniques use PCR to identify other diseases, so PCR and thermocyclers have many uses for detecting many diseases!
HERE'S THE CATCH:
Thermocyclers are the most expensive piece of equipment in the whole genetic disease identification toolbox. By contrast, the chemicals used in a single test cost just a few dollars. Thermocyclers can cost $10,000-$20,000 U.S. dollars. Most rely on uninterrupted electricity. If they fail or need maintenance in a foreign country, it often needs to be sent overseas-- which is very expensive. Cost is a big issue.
If thermocyclers could be made for, say, $100-$150, and be designed simple enough to repair locally with easily-to-obtain parts, and run on AC or battery electricity (and not reset when the power drops out), many more of them could be made and distributed to more remote areas, and not simply reside in larger, well-equipped cities. So while it's tempting to use the latest cutting edge technology, a very well-made low-cost design from inexpensive parts may be a better solution.
LOW-COST PCR FOR DISEASE DETECTION
My personal project choice of a thermocycler was motivated and inspired by Dr. Eva Harris, a Professor of Infectious Diseases at Berkeley who travels to developing nations to transfer technology to them. For over 17 years, she has had an amazing personal commitment to bringing science to nations such as Nicaragua that need it to fight Dengue Fever. She is incredibly inspiring and a great speaker, in touch with both the science and the people. Listen to her explain the frightening epidemic growth of Dengue and how it's spreading around the world-- even to the United States (Start on section #4, 6:05).
In her book, “A Low Cost Approach To PCR,” she emphasized the lack of thermocyclers in these areas and how they are needed for early and rapid disease detection.
Dr. Harris showed how to make PCR available to developing nations by replacing the thermocycler with boiling water and a manual lab process. She has already implemented this in locations around Central America. However, this manual procedure is cumbersome and requires constant attention, making it error prone.
LOW COST DETECTION SYSTEM
In order to utilize the capability of a low cost thermocycler, a low cost detection system must be paired with it. The detection system that I ultimately chose was a fluorescent detection system.
FLUORESCENT DNA DETECTION
One common method of analyzing amplified DNA is to integrate a fluorescent detection into the thermocycler. To put the mechanism of this detection simply, a small chemical binds to the DNA as its amplified throughout PCR and when a certain color of light is shined into the DNA, this chemical shines a unique color of light back. By measuring how much of this unique color is given off as the PCR progresses, it is possible to backtrack and find out the amount of base pairs of DNA that were present in the original sample. This capability allows for two types of detections. Firstly, this machine can detect the presence of simple diseases through the simple detection of increasing fluorescence throughout the reaction. Secondly, this machine can detect the presence of complex diseases by discovering the original amount of the targeted molecular material.
MY LOW-COST DISEASE DETECTION SOLUTION
I hypothesized that disease detection could be me made mobile, low cost, and low power with modern technology (microcontroller, resistors, gear systems, LCDs, etc.) if combined with intelligent math algorithms (PID advanced control algorithms) to fully automate Eva Harris's manual procedure.
My recent work with PCR in a Mayo laboratory with a mentor, my work with electronics and microcontrollers with my dad, and Dr. Harris' work all combined into an exciting and challenging project that stressed the importance of this machine and the tremendous benefit it could bring to these countries, potentially saving many lives.
WHAT IS A THERMOCYCLER?
A thermocycler is a machine used to amplify discreet strands of DNA. It raises and lowers temperatures, maintains temperatures for variable amounts of time with the process repeated multiple times. A thermocycler used in clinical settings to diagnose disease is often $10,000 or more, cost prohibitive for developing nations.
The purpose of my thermocycler is to inexpensively heat and cool a set of 0.5 ml test tubes containing a “master mix” of DNA fragments to be amplified. Cycling the mixture temperature between 3 target values and holding temperatures for specific dwell times doubles the DNA fragments with every full cycle. My thermocycler was validated against many criteria. Numerous small experiments tested temperature transitions required by PCR using digital thermometer measurements acquired by the microcontroller and monitored on an LCD display. The most difficult requirements were to raise test tube temperatures to a high target temperature of 95° C and to change temperatures (usually a by 20° C) in 20-30 seconds. Fast temperature changes (20-30 seconds) are required because some PCR temperature targets are held for as little as 30 seconds. Validating temperature transitions confirms that it met the most extreme PCR temperature targets (based on real virus laboratory tests) described in scientific manuals. My thermocycler met all thermal and key system requirements necessary for developing nations: inexpensive equipment; simplistic and easily maintainable with materials available worldwide. It will work in remote areas with unreliable electrical power; it is “off the grid”, can operate on rechargeable, solar-powered batteries; it is easily programmed and usable without complex training requirements.
WHAT HAVE I LEARNED?
I have learned so much about science (thermodynamics principles, methodical experimental design), technology (electrical circuits, microcontroller programming, LCDs, servos, pumps, temperature sensors, transistor switches), engineering (designing, debugging and redesigning to meet technical and system requirements) and math (deriving and using water mixing equations and feedback control loops). I learned that these areas are not distinct since they work together as parts to support the whole project. At first, everything didn’t work as expected-- fat grafting was much harder than I ever imagined.
My first engineering design had to be scrapped and re-designed due to excessive thermal losses. However, I didn’t give up; I kept nearly all of the electronics and math algorithms and made the physical and thermal design much better than the first! I am very excited about my progress, and have now completed a third design that is smaller, cheaper, more power efficient, and easier to build than the other prototypes.
Although I was the only student on this project, many people helped me; some helped directly on this project, while others helped me gain skills needed for the project, long before I even started. Dr. Chris Pierret inspired me with genetics projects for over two years, teaching me (in a Mayo Clinic Laboratory) how to extract DNA and analyze zebrafish. Stephanie Westcot, a graduate student in the same lab, taught me how to perform electrophoresis to analyze DNA. She taught me about PCR and supervised me while I performed my baseline PCR that I hope to use to validate my thermocycler. My dad, an electrical engineer, helped me learn electronics and taught me how to program microcontrollers since first grade. He also taught me how to combine microcontrollers with electronics. I used these programming and engineering skills when developing my thermocycler at home. My experiences in lego robotics helped improve my programming skills. My mom, a biologist and epidemiologist, taught me how to analyze data and make scientific presentations. She also helped me with my technical writing for this project. This year, I had my first peer-reviewed article published (Mathematical Modeling of Zebrafish Development, Zebrafish, Volume 9, Issue 4). I improved my public speaking skills by making poster presentations for 2 consecutive years at the 9th and 10th International Zebrafish Conference in Rochester, MN and Madison, WI.