Difference between revisions of "Team:UMaryland/Design"

  <p> The PCR machine is a common machine used in biological laboratories to amplify or extend fragments of DNA to be used in subsequent experiments. This tool is especially relevant to iGEM and SynBio labs who pave the way to vaster applications of We began this project with the vision to create a machine that would be

<p>Our first design for a DIY PCR machine was modeled after a more conventional PCR machine. This first prototype relied on two Peltier units stacked on top of each other to heat a customized aluminum block that held the PCR tubes. In order for the system to have feedback, we embedded a temperature sensor in the aluminum block to measure the temperature of the PCR tube wells. The sensor then reported back to an Arduino UNO, which then regulated the energy flow to the Peltier units, thereby regulating the temperature of the block and tubes. However, after much testing, this design proved to be unoriginal, expensive, and inefficient. While the conventionality of the design itself did not pose an issue, we realized that the parts used to assemble it were not as well-known or easily accessible to the general public, which we felt would take away from the possible applications of this machine. In addition, although the price of this first prototype was relatively inexpensive in contrast to laboratory grade PCR machines, the price still ranged in the hundreds of dollars. Finally, the greatest issue with our design was the inefficiency of the hardware; we found that the Peltier units were not able to quickly cycle through the desired temperatures, causing the unit to take 5 to 10 minutes just to rise up to 95℃. After considering all of these factors, we began a redesign of our machine to better suit the needs of the DIY market.</p>

<p> The idea for our current thermocycler design first came into form when we found that our original prototype was not ramping up to the desired temperatures fast enough. We thus looked into other options such as the heating element in a hair dryer. We found that the hair dryer was able to reach very high temperatures—much higher than the desired maximum of 95℃ for PCR—in a matter of seconds. We then made a decision to suspend construction on the Peltier-centered thermocycler in order to see how successful we could be with making a rapid PCR machine out of a hair dryer. Before this decision, we took into consideration the danger of working with a hair dryer, failure due to uncertainty that the machine could be effectively controlled, and, on top of that, having less time to work on it. Nevertheless, we took the risk.</br><i>Please continue on to see the design of our machine.</i>

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Although the process of PCR, the necessary hardware needed to do it is relatively simple. While different templates, primers, and polymerases can be used, a thermocycler capable of maintaining temperatures between 4 and 95 degrees is absolutely required. Current thermocyclers cost thousands of dollars, which is often prohibitively expensive for a DIY Bio effort. Making the device cheaper would give more people accessibility to this valuable lab tool, enabling breakthrough research in more places around the globe.

After a lot of soldering and reworking the internal safety measures inside the hairdryer, we were able to wire the system so that we could turn the heat on and off while running the fan continuously. Using tape, we secured a sheet of aluminium foil to the top of the heating unit of the hairdryer. The outer casing of the hairdryer had been removed. We placed a heat sensor inside the tin to measure the temperature of the air inside the machine.  By wiring the heat sensor to the Arduino we were able to receive input/feedback from the sensor and adjust heating of the device to maintain our desired setpoints. We were able to regulate the heat of the machine in order to produce proper thermocycling.

  <p>After construction of the can based cover we tried PCR once more and still found that the reaction did not occur. We assumed that the heat sensor might have been an issue,; the sensor was exposed to the moving air and was relaying information about the air temperature instead of the temperature inside of the PCR tubes. This meant that our feedback system was not accurately responding and controlling the temperature inside of the PCR tubes. Assuming the temperatures inside the machine were not representative of the temperatures inside the PCR tubes, we put the heat sensor inside a PCR tube with mineral oil and placed this inside one of the holes. We ran another PCR reaction, ran the products on a gel and saw a large band of the correct size, indicating that our machine had worked.

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<p style="text-align:center;font-size:32px;font-family: Tahoma, Geneva, sans-serif;"><b>Hardware</b>

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<img src="https://static.igem.org/mediawiki/2015/9/9f/UMD_PCR_hardware.jpg" style="width:1000px;height:558px;">

  The relays convert the low wattage outputs of the Arduino into a high wattage output needed to power the hairdryer. The relays are switches that can be triggered by the milliwatt output of the Arduino and can handle the 1.8 kilowatt power of the hairdryer.  

<b>Closing the loop.</b>With both the temperature sensor and the relays we are able to provide the micro-controller with the ability to regulate and cycle the machine at various temperatures. To allow for tight temperature regulation within the machine a proportional integral derivative control scheme was adopted. This scheme enabled the controller to take temperature readings and calculate rate at which the temperature is increasing, the constant error of the machine found through the integral term, and the proportional error which compares current temperature to a set point. The way our code is designed and implemented utilizes three setpoints, 95,70, and 50 degrees C, all of these are variable and able to be adjusted but for convince we will define the three with these set of temperature values. A any given time only one of these setpoints is active, and the PID control scheme regulates temperature at that specific value. Since the machine needs to cycle and hit at least 3 different temperatures our code also logs time after each setpoint is hit, thus allowing us to define a time interval after which the setpoint is altered. What this means is that if we define the first setpoint to be 95 degrees C that our code will execute and tell the machine to heat to 95 and once that temperature is reached it will trigger a timing function which after a defined period will reset the setpoint to 50 degrees which will then force the machine to cool down to the new setpoint.                   

<img src="https://static.igem.org/mediawiki/2015/d/d5/IGEMCYCDATAGRAPH.png" style="width:800px;height:400px;">

<img src="https://static.igem.org/mediawiki/2015/e/e8/IGEMUMDPCR.png" style="width:450px;height:450px;float:left;">

<p> We have had one successful amplification with our machine however we understand that repeatability is a vital component of all lab work and currently we are attempting to make our device repeatable. From our early days of testing we found that peltier units were not powerful enough to enable PCR tube to reach 95 degrees. On the other hand, the fan and heating element of a cheap hairdryer provide a control scheme that enables for rapid cycling of temperature. We have found that developing a housing for the PCR tubes and enabling even heat distribution is challenging. We often have found that our temperature sensor and the pcr reaction tube are not at the same temperature and degree of difference is a delta of over 10 degrees Celsius. We are currently working of milling a block of aluminum with better and more consistent heat transfer properties, and modeling the heat transfer within the can. Our ambition is that this will enable better control of temperature within the device.