Difference between revisions of "Team:UMaryland/Design"

Revision as of 03:02, 19 September 2015

Background

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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

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.

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.
Please continue on to see the design of our machine.

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UMD DIY PCR

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Our first design for a DIY PCR machine was modeled after a more conventional PCR machine design. This first prototype consisted of two Peltier units stacked on top of each other that would then heat a customized aluminum block that sat on top of the two units and 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.

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Design

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We began by working out how to wire the hairdryer so that we could regulate the heating unit and the fan separately.
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.
At this point, we tried to perform our first PCR reaction. Unfortunately, we soon found that we had melted our tube. We learned that the machine had difficulty with evenly distributing the heat. To better distribute the heat, we removed our tinfoil lid and replaced it with with a cut soda can. This can was designed with evenly spaced holes enabling for better heat distribution. Although we did not and still have not modeled the heat transfer of between the can's surface and the convection heating generated by the hair dryer, we were able to experimentally conclude that the heat distribution was more even across the can than the tin foil.

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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@@ Line 125: / Line 125: @@
 background-color:red;
 danger:wow;
-height:100px;
+height:150px;
 top:-220px;
 text-align:center;
@@ Line 273: / Line 273: @@
 <font color="#000000">#include</font> <font color="#434f54">&lt;</font><b><font color="#d35400">LiquidCrystal</font></b><font color="#434f54">.</font><font color="#000000">h</font><font color="#434f54">&gt;</font>
-<font color="#434f54">//The PID functions by adjusting a certain output in order to &nbsp;</font>
+<font color="#7e7e7e">/*The PID functions by adjusting a certain output in order to &nbsp;</font><font color="#7e7e7e"> * minimize the error between two values, which are the setpoint </font><font color="#7e7e7e"> * and the input.</font><font color="#7e7e7e"> * The PID function itself creates a PID controller and takes </font><font color="#7e7e7e"> * five parameters:</font><font color="#7e7e7e"> * Input: The value that needs to be controlled</font><font color="#7e7e7e"> * Output: The value that the PID will adjust</font><font color="#7e7e7e"> * Setpoint: The value that the input will be maintained at</font><font color="#7e7e7e"> * Kp,Ki,KD: Parameters that will affect how the output is adjusted</font><font color="#7e7e7e"> * Direct: Defines which direction the output will proceed given an error</font><font color="#7e7e7e"> */</font>
-<font color="#434f54">// minimize the error between two values, which are the setpoint </font>
-<font color="#434f54">// and the input.</font>
-<font color="#434f54">// The PID function itself creates a PID controller and takes </font>
-<font color="#434f54">// five parameters:</font>
-<font color="#434f54">// Input: The value that needs to be controlled</font>
-<font color="#434f54">// Output: The value that the PID will adjust</font>
-<font color="#434f54">// Setpoint: The value that the input will be maintained at</font>
-<font color="#434f54">// Kp,Ki,KD: Parameters that will affect how the output is adjusted</font>
-<font color="#434f54">// Direct: Defines which direction the output will proceed given an error</font>
 <font color="#434f54">// Define PID varaibles</font>
@@ Line 373: / Line 363: @@
 <font color="#000000">{</font>
   &nbsp;<font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">stepnow</font> <font color="#434f54">&lt;</font> <font color="#000000">cyclenum</font><font color="#000000">)</font> <font color="#000000">{</font>
-  &nbsp;&nbsp;&nbsp;<font color="#434f54">//The following code is only implemented once as the first cycle of the PCR</font>
+  &nbsp;&nbsp;&nbsp;<font color="#7e7e7e">/**The following code is only implemented once as the first cycle of the PCR</font><font color="#7e7e7e"> &nbsp;&nbsp;&nbsp;&nbsp;* This allows for the user to set conditions which may be different from the subsequent </font><font color="#7e7e7e"> &nbsp;&nbsp;&nbsp;&nbsp;* cycles. The code, however, works in much the same way as the other cycles</font><font color="#7e7e7e"> &nbsp;&nbsp;&nbsp;&nbsp;* at the most basic level. It divides each cycle into subcycles: activation, pcr and extension</font><font color="#7e7e7e"> &nbsp;&nbsp;&nbsp;&nbsp;* and enters each step based on stepnow(which denotes how many subcyles the program has entered) divisiblity by 3. </font><font color="#7e7e7e"> &nbsp;&nbsp;&nbsp;&nbsp;* The remainder for activation will always be 1, </font><font color="#7e7e7e"> &nbsp;&nbsp;&nbsp;&nbsp;* the remainder for pcr will be 2 and the remainder for extension will be 3</font><font color="#7e7e7e"> &nbsp;&nbsp;&nbsp;&nbsp;*/</font>
- &nbsp;&nbsp;&nbsp;<font color="#434f54">// This allows for the user to set conditions which may be different from the subsequent </font>
- &nbsp;&nbsp;&nbsp;<font color="#434f54">// cycles. The code, however, works in much the same way as the other cycles</font>
- &nbsp;&nbsp;&nbsp;<font color="#434f54">// at the most basic level. It divides each cycle into subcycles: activation, pcr and extension</font>
- &nbsp;&nbsp;&nbsp;<font color="#434f54">// and enters each step based on stepnow(which denotes how many subcyles the program has entered) divisiblity by 3. </font>
- &nbsp;&nbsp;&nbsp;<font color="#434f54">// The remainder for activation will always be 1, </font>
- &nbsp;&nbsp;&nbsp;<font color="#434f54">// the remainder for pcr will be 2 and the remainder for extension will be 3</font>
- &nbsp;&nbsp;&nbsp;&nbsp;
   &nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//Begins activation of the PCR by checking that this is the first cycle and that the current subcycle is activation </font>
   &nbsp;&nbsp;&nbsp;<font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#000000">(</font><font color="#000000">stepnow</font> <font color="#434f54">%</font> <font color="#000000">3</font><font color="#000000">)</font> <font color="#434f54">==</font> <font color="#000000">1</font><font color="#000000">)</font> <font color="#434f54">&&</font> <font color="#000000">(</font><font color="#000000">cycle</font> <font color="#434f54">==</font> <font color="#000000">2</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#000000">{</font>
@@ Line 394: / Line 377: @@
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">laststep</font><font color="#434f54">++</font><font color="#000000">;</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
-  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//If the pcr has maintained the setpoint for the desired amount </font>
+  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//If the pcr has maintained the setpoint for the desired amount of time, it will enter this loop and begin cooling</font>
- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//of time, it will enter this loop and begin cooling</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#d35400">millis</font><font color="#000000">(</font><font color="#000000">)</font> <font color="#434f54">&gt;</font> <font color="#000000">(</font><font color="#000000">t1Melt</font> <font color="#434f54">+</font> <font color="#000000">cycleStart</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#434f54">&&</font> <font color="#000000">(</font><font color="#000000">laststep</font> <font color="#434f54">==</font> <font color="#000000">stepnow</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">stepnow</font><font color="#434f54">++</font><font color="#000000">;</font>
@@ Line 414: / Line 396: @@
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">laststep</font><font color="#434f54">++</font><font color="#000000">;</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
-  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//checks if the machine has maintained the temperature for </font>
+  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//checks if the machine has maintained the temperature for the desired amount of time, and begins the next step</font>
- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//the desired amount of time, and begins the next step</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#d35400">millis</font><font color="#000000">(</font><font color="#000000">)</font> <font color="#434f54">&gt;</font> <font color="#000000">(</font><font color="#000000">t1PCR</font> <font color="#434f54">+</font> <font color="#000000">cycleStart</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#434f54">&&</font> <font color="#000000">(</font><font color="#000000">laststep</font> <font color="#434f54">==</font> <font color="#000000">stepnow</font><font color="#000000">)</font> <font color="#434f54">&&</font> <font color="#000000">(</font><font color="#000000">cycle</font> <font color="#434f54">==</font> <font color="#000000">2</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">stepnow</font><font color="#434f54">++</font><font color="#000000">;</font>
@@ Line 433: / Line 414: @@
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">laststep</font><font color="#434f54">++</font><font color="#000000">;</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
-  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//checks if the machine has maintained the temperature </font>
+  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">//checks if the machine has maintained the temperature for the desired amount of time, and begins the next step</font>
- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#434f54">// for the desired amount of time, and begins the next step</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#d35400">millis</font><font color="#000000">(</font><font color="#000000">)</font> <font color="#434f54">&gt;</font> <font color="#000000">(</font><font color="#000000">textension1</font> <font color="#434f54">+</font> <font color="#000000">cycleStart</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#434f54">&&</font> <font color="#000000">(</font><font color="#000000">laststep</font> <font color="#434f54">==</font> <font color="#000000">stepnow</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">pcr</font> <font color="#434f54">=</font> <font color="#00979c">"heating to melt "</font><font color="#000000">;</font>
@@ Line 443: / Line 423: @@
   &nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
+<font color="#7e7e7e">/**</font><font color="#7e7e7e"> * Begins the intermediate steps which work in an identical manner to the startup cycle </font><font color="#7e7e7e"> * and may differ only in the alloted temperatures and times for each subcycle</font><font color="#7e7e7e"> */</font>
- <font color="#434f54">//Begins the intermediate steps which work in an identical manner to the startup cycle </font>
- <font color="#434f54">//and may differ only in the alloted temperatures and times for each subcycle</font>
   &nbsp;&nbsp;&nbsp;<font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#000000">(</font><font color="#000000">stepnow</font> <font color="#434f54">%</font> <font color="#000000">3</font><font color="#000000">)</font> <font color="#434f54">==</font> <font color="#000000">1</font><font color="#000000">)</font> <font color="#434f54">&&</font> <font color="#000000">(</font><font color="#000000">cycle</font> <font color="#434f54">!</font><font color="#434f54">=</font> <font color="#000000">2</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">Setpoint</font> <font color="#434f54">=</font> <font color="#000000">meltT</font><font color="#000000">;</font>
@@ Line 491: / Line 468: @@
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
   &nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
- &nbsp;
+  &nbsp;<font color="#7e7e7e">/*</font><font color="#7e7e7e"> &nbsp;&nbsp;* Begins the last step, which again works in a similar fashion to the other steps, but may differ in the </font><font color="#7e7e7e"> &nbsp;&nbsp;* final extension temperature</font><font color="#7e7e7e"> &nbsp;&nbsp;*/</font>
-  &nbsp;<font color="#434f54">// Begins the last step, which again works in a similar fashion </font>
- &nbsp;<font color="#434f54">// to the other steps, but may differ in the final extension temperature</font>
- &nbsp;&nbsp;
   &nbsp;<font color="#000000">}</font> <font color="#5e6d03">else</font> <font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#000000">stepnow</font> <font color="#434f54">&gt;</font><font color="#434f54">=</font> <font color="#000000">cyclenum</font> <font color="#434f54">&&</font> <font color="#000000">stepnow</font> <font color="#434f54">&lt;</font><font color="#434f54">=</font> <font color="#000000">cyclenum</font> <font color="#434f54">+</font> <font color="#000000">2</font> <font color="#000000">)</font><font color="#000000">)</font> <font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;<font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#000000">(</font><font color="#000000">stepnow</font> <font color="#434f54">%</font> <font color="#000000">3</font><font color="#000000">)</font> <font color="#434f54">==</font> <font color="#000000">1</font><font color="#000000">)</font><font color="#000000">)</font> <font color="#000000">{</font>
@@ Line 541: / Line 515: @@
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
   &nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
-  &nbsp;
+  &nbsp;<font color="#7e7e7e">/**</font><font color="#7e7e7e"> &nbsp;&nbsp;* Once the last step has been completed, determined by stepnow being greater than cyclenum+2,</font><font color="#7e7e7e"> &nbsp;&nbsp;* the code will tell the pcr to hold at 4 C. It will continue looping at this step indefinitely</font><font color="#7e7e7e"> &nbsp;&nbsp;* because the conditions will no longer satisfy any of the other if statement</font><font color="#7e7e7e"> &nbsp;&nbsp;*/</font>
- &nbsp;&nbsp;<font color="#434f54">// Once the last step has been completed, determined by </font>
- &nbsp;&nbsp;<font color="#434f54">// stepnow being greater than cyclenum+2,the code will tell the </font>
- &nbsp;&nbsp;<font color="#434f54">// pcr to hold at 4 C. It will continue looping at this step indefinitely</font>
- &nbsp;&nbsp;<font color="#434f54">// because the conditions will no longer satisfy any of the other if statement</font>
- &nbsp;&nbsp;
   &nbsp;<font color="#000000">}</font> <font color="#5e6d03">else</font> <font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;<font color="#00979c">int</font> <font color="#000000">strt</font> <font color="#434f54">=</font> <font color="#000000">1</font><font color="#000000">;</font>
@@ Line 561: / Line 530: @@
   &nbsp;&nbsp;&nbsp;<font color="#000000">}</font>
-  &nbsp;&nbsp;&nbsp;<font color="#434f54">//Once the pcr has reached 37 C, the pcr will </font>
+  &nbsp;&nbsp;&nbsp;<font color="#434f54">//Once the pcr has reached 37 C, the pcr will print out that the pcr has shutt off</font>
- &nbsp;&nbsp;&nbsp;<font color="#434f54">//print out that the pcr has shutt off</font>
   &nbsp;&nbsp;&nbsp;<font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#000000">Input</font> <font color="#434f54">-</font> <font color="#000000">38</font> <font color="#434f54">+</font> <font color="#000000">1</font><font color="#000000">)</font> <font color="#434f54">&lt;</font> <font color="#000000">0</font><font color="#000000">)</font> <font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;&nbsp;<font color="#d35400">digitalWrite</font><font color="#000000">(</font><font color="#000000">signalr1</font><font color="#434f54">,</font> <font color="#00979c">HIGH</font><font color="#000000">)</font><font color="#000000">;</font>
@@ Line 585: / Line 553: @@
   &nbsp;&nbsp;&nbsp;<font color="#d35400">digitalWrite</font><font color="#000000">(</font><font color="#000000">signalr2</font><font color="#434f54">,</font> <font color="#00979c">LOW</font><font color="#000000">)</font><font color="#000000">;</font>
   &nbsp;&nbsp;&nbsp;<b><font color="#d35400">Serial</font></b><font color="#434f54">.</font><font color="#d35400">println</font><font color="#000000">(</font><font color="#00979c">"COOLING"</font><font color="#000000">)</font><font color="#000000">;</font>
-  <font color="#434f54">// if the pcr machine's temperature reaches 97, cooling will </font>
+  <font color="#434f54">// if the pcr machine's temperature reaches 97, cooling will automatically be initiated and a death message will be displayed</font>
- <font color="#434f54">// &nbsp;&nbsp;automatically be initiated and a death message will be displayed</font>
   &nbsp;<font color="#000000">}</font><font color="#5e6d03">else</font> <font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">(</font><font color="#000000">Input</font> <font color="#434f54">-</font> <font color="#000000">deathT</font> <font color="#434f54">+</font><font color="#000000">1</font><font color="#000000">)</font> <font color="#434f54">&gt;</font> <font color="#000000">0</font><font color="#000000">)</font><font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;<font color="#d35400">digitalWrite</font><font color="#000000">(</font><font color="#000000">signalr1</font><font color="#434f54">,</font> <font color="#00979c">HIGH</font><font color="#000000">)</font><font color="#000000">;</font>
@@ Line 592: / Line 559: @@
   &nbsp;&nbsp;&nbsp;<b><font color="#d35400">Serial</font></b><font color="#434f54">.</font><font color="#d35400">println</font><font color="#000000">(</font><font color="#00979c">"DEATH"</font><font color="#000000">)</font><font color="#000000">;</font>
-<font color="#434f54">// these two statements work on the maintaining the temperature, by </font>
+<font color="#7e7e7e">/* these two statements work on the maintaining the temperature, by controlling heating and cooling when there is overshoot or undershoot </font><font color="#7e7e7e"> &nbsp;&nbsp;in the temperature*/</font>
-<font color="#434f54">// controlling heating and cooling when there is overshoot or undershoot </font>
-<font color="#434f54">// in the temperature</font>
   &nbsp;<font color="#000000">}</font> <font color="#5e6d03">else</font> <font color="#5e6d03">if</font> <font color="#000000">(</font><font color="#000000">Output</font> <font color="#434f54">&gt;</font> <font color="#000000">now</font> <font color="#434f54">-</font> <font color="#000000">windowStartTime</font> <font color="#434f54">-</font> <font color="#000000">1</font><font color="#000000">)</font> <font color="#000000">{</font>
   &nbsp;&nbsp;&nbsp;<font color="#d35400">digitalWrite</font><font color="#000000">(</font><font color="#000000">signalr1</font><font color="#434f54">,</font> <font color="#00979c">HIGH</font><font color="#000000">)</font><font color="#000000">;</font>