Team:TU Darmstadt/result/electrical engineering

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<a href="https://2013.igem.org/Team:TU_Darmstadt/result/molecular_engineering">
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<font size="8" color="#F0F8FF" face="Arial regular">Molecular engineering |</font>
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</a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/result/electrical_engineering">
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<font size="8" color="#F0F8FF" face="Arial regular">Electrical engineering </font>
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<h1 align="center"><font size="6" color="#F0F8FF" face="Arial regular">Electrical engineering</font></h1>
<h1 align="center"><font size="6" color="#F0F8FF" face="Arial regular">Electrical engineering</font></h1>
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<br>
 
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<font size="4" color="#F0F8FF" face="Arial regular">
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<b>The Handheld:</b><br>
 
<p text-aligne:left style="margin-left:50px; margin-right:50px">
<p text-aligne:left style="margin-left:50px; margin-right:50px">
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blabblabalalbal <sup><span style="color:blue">[1]</span></sup> showed that LSSmOrange has an excitation maximum by
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<b>Handheld development:</b>
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blablabla
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</p>
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<br><br>
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<center><img alt="Spectra LSSmOrange" src="/wiki/images/a/af/LSSmOrange_Diagramm.png" width="800" height="400"></center> <br>
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<font size="3" color="#F0F8FF" face="Arial regular">
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<center>Graph 1: "BL21DE3pLys[pBR-IBA2-LSSmOrange]"<br>
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Excitation spectrum (dashed line) and emission spectrum (solid line) from
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LSSmOrange with marked maximums.<br><br></center><br><br>
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<br><br>
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<center><img alt="LSSmOrange bac" src="/wiki/images/a/a7/Lssmorangepicbact.png
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" width="400" height="500"></center> <br>
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<center>Graph 1.1: "BL21DE3pLys[pBR-IBA2-LSSmOrange]":<br>
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Pellet of E.coli Bl21(DE3) cells with expressed LSSmOrange. Left side: without UV radiation, right side: with UV radiation.<br><br></center><br><br>
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<br><br>
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<b>mKate:</b><br>
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
<p text-aligne:left style="margin-left:50px; margin-right:50px">
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mKate2 is a red fluorescent protein that we want to use as a acceptor for FRET. According to Evrogen <sup><span style="color:blue">[2]</span></sup>
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Our handheld contains an Arduino Uno Rev3 microcontroler which is connected to a HC-05 bluetooth module.Two LEDs are connected over 100 ohm resistances to digital ports on the Arduino Uno. One of the LEDs emitts radiation with a wavelength of 450 nm permanently during the detection process. The other LED emmits light of a wavelength at 575 nm but works as a photo diode. Sensing details are explained below.
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mKate2 has an excitation maximum at 588 nm and an emission maximum at 633 nm. The
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<br>
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diagram below (graph 2, colored dots) shows that the excitation maximum is at 579 nm and the
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Favorable for light intensity measurements, however, is to operate the LED in reverse direction. Here it behaves like a capacitor with a parallel light-dependent current source. The anode is connected to ground and the cathode is connected to an IO port pin of the arduino microcontroller.
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emission maximum is 611 nm. The first peak does not look exactly like a single peak, but we can see a
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<br>
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clear maximum. The problem in our FRET system is the very low intensity which results
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The measurement is done in 3 steps:
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in the next diagram (graph 3) in a huge difference of intensity of the two fluorescence
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<br>
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proteins.<br><br>
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1. LED charging by switching the pins on high and wait.<br>
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2.Moving the pins in high-impedance input (no pull-up!). The current now flows from the capacitor very slowly via the current source to GND. Therefore, the voltage drops steadily and drops so far that the microcontroller recognizes it as low.<br>
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3. The microcontroller measures the time, when the input falls to LOW
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<br>
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The shorter the time measured, the higher is the light intensity.
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<br>
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<br>
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</p>
 
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<center><img alt="Spectra mKate" src="/wiki/images/f/fa/Spectra_mKate.png" width="800" height="400"></center> <br>
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<br>
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<center>Graph 2: "BL21DE3pLys[pBR-IBA2-mKate]":<br>
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<font size="3" color="#F0F8FF" face="Arial regular">
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Excitation spectrum (dashed line) and emission spectrum (solid line) of
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mKate with marked maximums<br><br></center><br><br>
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<br><br>
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<center><img alt="mKate bac" src="/wiki/images/3/3f/Mkatepicbact.png" width="400" height="500"></center> <br>
 
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<center>Graph 2.1: "BL21DE3pLys[pBR-IBA2-mKate]":<br>
 
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Pellet of E.coli Bl21(DE3) cells with expressed mKate. Left side: without UV radiation, right side: with UV radiation.<br><br></center><br><br>
 
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<br><br>
 
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<b>FRET:</b><br>
 
<p text-aligne:left style="margin-left:50px; margin-right:50px">
<p text-aligne:left style="margin-left:50px; margin-right:50px">
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Both diagrams in an overlay shows us the FRET system. LSSmOrange is the donor and mKate is the
 
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acceptor. For an optimum in FRET, it should display a stronger overlap between the emission maximum of LSSmOrange and the excitation maximum of mKate.<br><br>
 
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</p>
 
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<center><img alt="Spectra FRET" src="/wiki/images/4/41/FRET_Normalized_2.png" width="800" height="400"></center> <br>
 
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<center>Graph 3: "FRET pair LSSmOrange-mKate":<br>
 
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Normalized excitation and emission spectra overlay of of graph 1 and graph 2.<br><br></center><br><br>
 
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<br>
 
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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You can find our arduino code, open soure on the folowing site:
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The next figure is from the paper of M. Shcherbakova. In this figure one can
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<a href=" http://github.com/BastianWagner/Mycotoxin-Arduino-Handheld/blob/master/handheld_arduino_code.ino
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clearly see the overlap between donor and acceptor in an optimal FRET system. <br><br>
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">Mycotoxin Handheld Arduino code</a><br>
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</p>
 
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<center><img alt="Spectra" src="/wiki/images/6/6c/ShcherbakovaSpectra.png" width="400" height="349"></center> <br>
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</p>
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<center>Excitation and emission spectra overlay of donor LSSmOrange and acceptor mKate2 <sup><span style="color:blue">[1]</span></sup><br>
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<br><br>
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(dashed line = excitation spectrum, solid line = emission spectrum)</center><br><br>
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<img src="https://static.igem.org/mediawiki/2013/9/9b/Darmstadt13_elEng_handheld_Steckplatine.png" alt="" width="50%">
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<br><br><br>
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
<p text-aligne:left style="margin-left:50px; margin-right:50px">
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<b>LED Sensing:</b><br>
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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Light emitting diodes are robust, low-cost, and energy efficient. LEDs cover an increasingly broad spectral range from UV to near infrared.
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<center><b>Growth curves:</b></center>
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LEDs can be both light emitters and detectors. For example, an LED that emits greenish-yellow light at the peak wavelength of about 555 nm detects green light at the peak wavelength of about 525 nm and over the spectral width of 50 nm.
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<br>
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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Almost every LED is capable of detecting a relatively narrow band of wavelength, with different
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sensitivity. When an LED is subjected to light, it generates a backwards biased current, proportional to the light striking the diode, typical about 50pA.
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To determine the influence of the induced constructs mKate and LSSmOrange on the BL21DE3, growth curves were used. During a time period of 8 h the OD of the samples was measured at a wavelength of 600 nm in time steps of 15 min. The measurement was started at an OD<sub>600 nm</sub>  value of 0.3.  With the help of the following formulas the growth rate k and the doubling time t<sub>d</sub> were calculated:
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A precise measurement of the LED photocurrent is possible, using inherent capacitance of the diode itself (typically picoFarads) and  microcontroller I/O ports with configurable internal pull-ups states and built-in digital timer-counter.  
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</p>
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</p>
 
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<img alt="Spectra" src="/wiki/images/5/52/Johanna_Formel.JPG" width="200" height="100">
 
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<b>App development:</b><br>
<p text-aligne:left style="margin-left:50px; margin-right:50px">
<p text-aligne:left style="margin-left:50px; margin-right:50px">
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The calculated values of k and td of the competent cells BL21DE3 containing the mKate or LSSmOrange construct were compared to the level of the wild type strain, to reveal possible toxicity of the constructs. In the following diagrams the OD<sub>600</sub>, respectively the ln OD<sub>600</sub>, values are plotted against the time and in the table below the resulting values of the growth rates k and the doubling times td are listed. <br><br>
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Click here for our mycotoxin detector <a href="https://play.google.com/store/apps/details?id=de.tudarmstadt.se.igem&hl=de"> App </a>
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<center><img alt="Growth curve" src="/wiki/images/6/65/WACHSTUMSKURVE2.jpg" width="800" height="400"></center> <br>
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<br><br><br>
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<center>Graph 4: "Growth curves":<br>
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<center>Growth curves of BL21DE3 WT, BL21DE3 mKate and BL21DE3 LSSmOrange. The OD<sub>600</sub> values are plotted against the time [min]. </center><br><br>
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<br>
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<img alt="Choose Mykotoxine" src="https://static.igem.org/mediawiki/2013/8/8e/Screenshot_choose.png" width="200" height="400" align="left">  
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<center><img alt="Growth curve2" src="/wiki/images/a/a1/WACHSTUMSKURVE2-LOG.jpg" width="800" height="400"></center> <br>
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The App offers a choice of various mycotoxins and supplemantary information. Additionally, it features a graphical diagramme of the meassured data. It supports bluetooth as well as usb, and can connect to external databases on previous messurements and analyses.
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<center>Graph 5: "Growth curves log":<br>
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<center>Semi-logarithmic growth curves of BL21DE3 WT, BL21DE3 mKate and BL21DE3 LSSmOrange. The ln (OD<sub>600</sub> values) are plotted against the time [min].</center><br>
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<img alt="Plug" src="/wiki/images/e/ee/Screenshot_plug.png" width="200" height="400" align="right"><br>
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<br><br><br><br>
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<br><br>
 
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Table1: The growth rate k and the doubling time t<sub>d</sub> of BL21DE3 WT, BL21DE3 mKate and BL21DE3LSSmOrange.
 
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<!-- Tabelle -->  
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<b>Bluetooth communication:</b><br>
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For our handheld we use a HC-05 module from Frima LC Technology. This module is easy to connect with Arduino boards and, additionally, is low priced. The HC-05 module enables sending and receiving data from and to other devices via bluetooth.
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<table border="1">
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  <tr>
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    <th></th>
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<td><b>BL21DE3(pPR-IBA2)</b></td>
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<td><b>BL21DE3[pPR-IBA2-LSSmOrange]</b></td>
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    <td><b>BL21DE3[pPR-IBA2-mKate]</b></td>
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<th><b>growth rate µ [min-1]</b></th>
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    <td>0,0286</td>
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    <td>0,0287</td>
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    <td>0,0232</td>
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    <th><b>doubling time td [min]</b></th>
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    <td>24,2459</td>
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    <td>24,1514</td>
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    <td>29,877</td>
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<img alt="Bluetooth" src="/wiki/images/8/85/Hc-05.jpg" width="250" height="238" align="right"> <br>
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"Bluetooth device"
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As can be seen in the two diagrams the course of the growth curves are rather similar. The growth rate as well as the doubling time of the wild type BL21DE3 and the one including the LSSmOrange construct are almost identical. The growth rate of the strain including the mKate construct is slightly lower and therefore the doubling time is around 5 min higher. All in all, the differences of the samples including the constructs compared to the wild type BL21DE2 are negligibly low. Therefore it can be assumed that the construct mKate as well as LSSmOrange are not toxic for the BL21DE3 cells and have no or a negligibly low influence on the growth of the cells. 
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<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2></center>
<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2></center>
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<ol>
<ol>
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<li style="margin-left:15px; margin-right:50px; text-align:justify">Daria M. Shcherbakova et al. (2012) <i>An Orange Fluorescent Protein with a Large Stokes Shift for Single-Excitation Multicolor FCCS and FRET Imaging.</i> J. Am. Chem. Soc. 134 (18), 7913–7923</li>
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<li style="margin-left:15px; margin-right:50px; text-align:justify">G.W.Mitchell and J.W.Hastings: A Stable, Inexpensive, Solid-State Photomultiplier Photometer <br>
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Biological Laboratories, Havard University, Cambridge, Massachusetts 02138<br>
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Analytical Biochemistry 39, 243-250 (1971)<br></li>
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<li style="margin-left:15px; margin-right:50px; text-align:justify">Hanwen Yan: An Inexpensive LED-Based Fluorometer Used to Study a Hairpin-Based DNA Nanomachine<br>
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Staples High School, 70 North Avenue, Westport, CT 06880 USA<br></li>
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<li style="margin-left:15px; margin-right:50px; text-align:justify">Nuts and Volts – November 2007<br></li>
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<li style="margin-left:15px; margin-right:50px; text-align:justify">Nuts and Volts – Mai  2013<br></li>
 +
<li style="margin-left:15px; margin-right:50px; text-align:justify">Forrest M. Mims III: Sun photometer with light-emitting diodes as spectrally selective detectors<br>
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The author is with Science Probe, Inc., 433 Twin Oak Road, Seguin, Texas 78155. Received 21 February 1992. © 1992 Optical Society of America<br></li>
 +
 
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<li style="margin-left:15px; margin-right:50px; text-align:justify">W J O’Hagan et al.: MHz LED source for nanosecond fluorescence sensing<br>
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Meas. Sci. Technol. 13 (2002) 84–91; Department of Physics and Applied Physics, University of Strathclyde, Glasgow G4 0NG, UK<br></li>
 +
 
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<li style="margin-left:15px; margin-right:50px; text-align:justify">Andrew E. Moe: Improvements in LED-based fluorescence analysis systems<br>
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Department of Electrical Engineering, University of Washington, Seattle, WA 98195-2500, USA<br></li>
 +
 
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<li style="margin-left:15px; margin-right:50px; text-align:justify">Radovan Stojanovic and Dejan Karadaglic: An optical sensing approach based on light emitting diodes<br>Journal of Physics: Conference Series 76 (2007) 012054<br></li>
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<li style="margin-left:15px; margin-right:50px; text-align:justify">http://www.evrogen.com/products/basicFPs.shtml</li>
 
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[1] J Am Chem Soc. 2012 May 9; 134(18): 7913–7923. doi:10.1021/ja3018972
 
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Daria M. Shcherbakova, An orange fluorescent protein with a large Stokes shift for
 
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single-excitation multicolor FCCS and FRET imaging.<br>
 
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[2] http://www.evrogen.com/products/basicFPs.shtml -->
 
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Latest revision as of 03:53, 5 October 2013







Molecular engineering | Electrical engineering





Electrical engineering

Handheld development:

Our handheld contains an Arduino Uno Rev3 microcontroler which is connected to a HC-05 bluetooth module.Two LEDs are connected over 100 ohm resistances to digital ports on the Arduino Uno. One of the LEDs emitts radiation with a wavelength of 450 nm permanently during the detection process. The other LED emmits light of a wavelength at 575 nm but works as a photo diode. Sensing details are explained below.
Favorable for light intensity measurements, however, is to operate the LED in reverse direction. Here it behaves like a capacitor with a parallel light-dependent current source. The anode is connected to ground and the cathode is connected to an IO port pin of the arduino microcontroller.
The measurement is done in 3 steps:
1. LED charging by switching the pins on high and wait.
2.Moving the pins in high-impedance input (no pull-up!). The current now flows from the capacitor very slowly via the current source to GND. Therefore, the voltage drops steadily and drops so far that the microcontroller recognizes it as low.
3. The microcontroller measures the time, when the input falls to LOW
The shorter the time measured, the higher is the light intensity.


You can find our arduino code, open soure on the folowing site: Mycotoxin Handheld Arduino code







LED Sensing:

Light emitting diodes are robust, low-cost, and energy efficient. LEDs cover an increasingly broad spectral range from UV to near infrared. LEDs can be both light emitters and detectors. For example, an LED that emits greenish-yellow light at the peak wavelength of about 555 nm detects green light at the peak wavelength of about 525 nm and over the spectral width of 50 nm. Almost every LED is capable of detecting a relatively narrow band of wavelength, with different sensitivity. When an LED is subjected to light, it generates a backwards biased current, proportional to the light striking the diode, typical about 50pA. A precise measurement of the LED photocurrent is possible, using inherent capacitance of the diode itself (typically picoFarads) and microcontroller I/O ports with configurable internal pull-ups states and built-in digital timer-counter.



App development:

Click here for our mycotoxin detector App


Choose Mykotoxine The App offers a choice of various mycotoxins and supplemantary information. Additionally, it features a graphical diagramme of the meassured data. It supports bluetooth as well as usb, and can connect to external databases on previous messurements and analyses. Plug
















Bluetooth communication:

For our handheld we use a HC-05 module from Frima LC Technology. This module is easy to connect with Arduino boards and, additionally, is low priced. The HC-05 module enables sending and receiving data from and to other devices via bluetooth. Bluetooth
"Bluetooth device"










References

  1. G.W.Mitchell and J.W.Hastings: A Stable, Inexpensive, Solid-State Photomultiplier Photometer
    Biological Laboratories, Havard University, Cambridge, Massachusetts 02138
    Analytical Biochemistry 39, 243-250 (1971)
  2. Hanwen Yan: An Inexpensive LED-Based Fluorometer Used to Study a Hairpin-Based DNA Nanomachine
    Staples High School, 70 North Avenue, Westport, CT 06880 USA
  3. Nuts and Volts – November 2007
  4. Nuts and Volts – Mai 2013
  5. Forrest M. Mims III: Sun photometer with light-emitting diodes as spectrally selective detectors
    The author is with Science Probe, Inc., 433 Twin Oak Road, Seguin, Texas 78155. Received 21 February 1992. © 1992 Optical Society of America
  6. W J O’Hagan et al.: MHz LED source for nanosecond fluorescence sensing
    Meas. Sci. Technol. 13 (2002) 84–91; Department of Physics and Applied Physics, University of Strathclyde, Glasgow G4 0NG, UK
  7. Andrew E. Moe: Improvements in LED-based fluorescence analysis systems
    Department of Electrical Engineering, University of Washington, Seattle, WA 98195-2500, USA
  8. Radovan Stojanovic and Dejan Karadaglic: An optical sensing approach based on light emitting diodes
    Journal of Physics: Conference Series 76 (2007) 012054