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<img id="TopPicture" width="960" src="https://static.igem.org/mediawiki/2017/f/fc/T--Munich--FrontPagePicture_Hardware.jpg"> | <img id="TopPicture" width="960" src="https://static.igem.org/mediawiki/2017/f/fc/T--Munich--FrontPagePicture_Hardware.jpg"> | ||
+ | <div> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/7/71/T--Munich--Prophecy.svg" height=50> | ||
+ | </div> | ||
+ | <audio controls> | ||
+ | <source src="https://static.igem.org/mediawiki/2017/8/87/Lightbringer_Prophecy.mp4" type="audio/mpeg"> | ||
+ | </audio> | ||
<table width="960" border=0 cellspacing=0 cellpadding=10> | <table width="960" border=0 cellspacing=0 cellpadding=10> | ||
<tr> | <tr> | ||
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Our pathogen detection approach relies on Cas13a digesting RNA. A common way of monitoring RNase activities is using commercially available RNaseAlert, consisting of a fluorescent RNA beacon. This is impractical for in-field applications because commercial fluorescence detectors are expensive and inconveniently large. We therefore make our pathogen detection system fit for in-field applications by developing a cheap and handy fluorescence detector. Although many previous iGEM teams constructed fluorescence detectors, we could not find any that had a high enough sensitivity or the ability to measure fluorescence quantitatively. We therefore constructed a detector matching our requirements and compared it to others in a cost vs sensitivity diagram. | Our pathogen detection approach relies on Cas13a digesting RNA. A common way of monitoring RNase activities is using commercially available RNaseAlert, consisting of a fluorescent RNA beacon. This is impractical for in-field applications because commercial fluorescence detectors are expensive and inconveniently large. We therefore make our pathogen detection system fit for in-field applications by developing a cheap and handy fluorescence detector. Although many previous iGEM teams constructed fluorescence detectors, we could not find any that had a high enough sensitivity or the ability to measure fluorescence quantitatively. We therefore constructed a detector matching our requirements and compared it to others in a cost vs sensitivity diagram. | ||
</p> | </p> | ||
+ | |||
+ | <div class="captionPicture"> | ||
+ | <img width=678 src="https://static.igem.org/mediawiki/2017/a/a4/T--Munich--Hardware_costvssensetivity.svg"> | ||
+ | <p> | ||
+ | Cost vs. sensitivity diagram of several fluorescence detectors. We compared commercially available detectors (orange dots), low-cost detectors from publications<sup><a class="myLink" href="#ref_1">1-5</a></sup> (green dots) and detectors from other iGEM teams (blue dots) to our fluorescence detector (red dot). | ||
+ | </p> | ||
+ | </div> | ||
<p class="introduction"> | <p class="introduction"> | ||
Our detector is paper-based and can detect fluorescein concentrations down to 100 nM. The detector is able to automatically | Our detector is paper-based and can detect fluorescein concentrations down to 100 nM. The detector is able to automatically | ||
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</p> | </p> | ||
<div class="captionPicture"> | <div class="captionPicture"> | ||
− | <img src="https://static.igem.org/mediawiki/2017/e/e2/T--Munich--Hardware_kinetic.png"> | + | <img id="sensitivity" src="https://static.igem.org/mediawiki/2017/e/e2/T--Munich--Hardware_kinetic.png"> |
<p> | <p> | ||
Time lapse measurement of Cas13a digesting RNaseAlert on paper using our detector. The | Time lapse measurement of Cas13a digesting RNaseAlert on paper using our detector. The | ||
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</div> | </div> | ||
<p class="introduction"> | <p class="introduction"> | ||
− | The time traces show an enzymatic reaction taking place on filter paper. For our experiments we used 185 nM RNaseAlert. By assuming that RNase A digested all RNaseAlert, we conclude that 185 nM of RNaseAlert have an equivalent fluorescence to | + | The time traces show an enzymatic reaction taking place on filter paper. For our experiments we used 185 nM RNaseAlert. By assuming that RNase A digested all RNaseAlert, we conclude that 185 nM of RNaseAlert have an equivalent fluorescence to 10 µM fluorescein. Our detection limit for RNaseAlert is therefore around 50 times lower which corresponds to a RNaseAlert concentration lower than 10 nM. This proves that our detector is sensitive |
enough and meets our requirements. However the detector is not limited to our specific application but can be used | enough and meets our requirements. However the detector is not limited to our specific application but can be used | ||
for the detection of any fluorescence signal in biological or chemical systems. We therefore think that our detector | for the detection of any fluorescence signal in biological or chemical systems. We therefore think that our detector | ||
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<p> | <p> | ||
Transmission spectra of the chosen filter foils of our detector. The transmission spectra for the | Transmission spectra of the chosen filter foils of our detector. The transmission spectra for the | ||
− | orange filter foil(orange graph) has nearly no overlap with the transmission spectra of the blue filter foil(blue graph). | + | orange filter foil (orange graph) has nearly no overlap with the transmission spectra of the blue filter foil (blue graph). |
− | The transmission spectra of two blue and one filter foil(black graph) is therefore nearly 0 up to 700 nm. | + | The transmission spectra of two blue and one filter foil (black graph) is therefore nearly 0 up to 700 nm. |
</p> | </p> | ||
</div> | </div> | ||
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<h4>3D Printed Parts</h4> | <h4>3D Printed Parts</h4> | ||
<p> | <p> | ||
− | We intended to put the LED,the fluorescence sample and the LDR in direct proximity, to ensures a maximum use | + | We intended to put the LED, the fluorescence sample and the LDR in direct proximity, to ensures a maximum use |
of excitation light and emission light. We therefore chose a sandwich-like design for our sample holder that can be | of excitation light and emission light. We therefore chose a sandwich-like design for our sample holder that can be | ||
placed into a slot where the detection system snaps in and keeps the sample in position. | placed into a slot where the detection system snaps in and keeps the sample in position. | ||
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the detection windows because tape is usually autofluorescent and causes a high background signal. A piece of filter | the detection windows because tape is usually autofluorescent and causes a high background signal. A piece of filter | ||
paper is placed between the two halves of the sandwich. The upper and lower part of this sandwich are pressed | paper is placed between the two halves of the sandwich. The upper and lower part of this sandwich are pressed | ||
− | together with magnets to hold the filter paper in position and ensure an user-friendly exchange of filter papers. We also designed a <a class="myLink" href"https://2017.igem.org/Team:Munich/Hardware/Paperstrip"> | + | together with magnets to hold the filter paper in position and ensure an user-friendly exchange of filter papers. We also designed a <a class="myLink" href="https://2017.igem.org/Team:Munich/Hardware/Paperstrip">paper strip</a> that matches our sample holder, to provide a low-cost and durable platform to store a reaction mix. An |
explosion drawing of the sample holder is shown in the figure below. | explosion drawing of the sample holder is shown in the figure below. | ||
</p> | </p> | ||
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<p> | <p> | ||
The sandwich can now be inserted into the slot of the detection device. The LED and the LDR are mounted onto | The sandwich can now be inserted into the slot of the detection device. The LED and the LDR are mounted onto | ||
− | beams at opposite | + | beams at opposite sides of the detection device. The sandwich snaps in when the LED and the LDR are at the right |
position under the excitation and over detection window. Four magnets apply an additional force to the beams and | position under the excitation and over detection window. Four magnets apply an additional force to the beams and | ||
press the LED and the LDR close together. This design ensures that the distance between filter paper, LED and | press the LED and the LDR close together. This design ensures that the distance between filter paper, LED and | ||
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<img src="https://static.igem.org/mediawiki/2017/3/32/T--Munich--Hardware_explodetdetectiondev.svg" width="700"> | <img src="https://static.igem.org/mediawiki/2017/3/32/T--Munich--Hardware_explodetdetectiondev.svg" width="700"> | ||
<p> | <p> | ||
− | Explosion drawing of detection slot. | + | Explosion drawing of the detection slot. |
</p> | </p> | ||
</div> | </div> | ||
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<tr class="lastRow"><td colspan=6 align=center valign=center> | <tr class="lastRow"><td colspan=6 align=center valign=center> | ||
− | <h4>List of Materials and Cost Calculation</h4> | + | <h4 id="cost">List of Materials and Cost Calculation</h4> |
<table class="myTable" width=60%> | <table class="myTable" width=60%> | ||
<th class="leftAligned">Used item</th> | <th class="leftAligned">Used item</th> | ||
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</td> | </td> | ||
</tr> | </tr> | ||
+ | |||
+ | |||
<tr class="lastRow"><td colspan=6 align=center valign=center> | <tr class="lastRow"><td colspan=6 align=center valign=center> | ||
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<p> | <p> | ||
where <i>k</i> is a constant that depends on the transmission spectra of the filter foils, the spectra of the fluorophore, the | where <i>k</i> is a constant that depends on the transmission spectra of the filter foils, the spectra of the fluorophore, the | ||
− | spectra of the LED and light scattering effects of the filter paper,but not on <i>I<sub>0</sub></i>. <i>k</i> can be assumed to be constant for one specific measurement set-up. | + | spectra of the LED and light scattering effects of the filter paper, but not on <i>I<sub>0</sub></i>. <i>k</i> can be assumed to be constant for one specific measurement set-up. |
</p> | </p> | ||
<p id="equation16"> | <p id="equation16"> | ||
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We therefore prepared 10-fold dilutions of fluorescein from 100 nM to 1 mM. For each measurement we pipetted 30 µl of sample on | We therefore prepared 10-fold dilutions of fluorescein from 100 nM to 1 mM. For each measurement we pipetted 30 µl of sample on | ||
a fresh filter paper, placed it in the detector and turned on the LED. We measured the resistance <i>R<sub>LDR</sub></i> directly with a multimeter. After each measurement the detector was cleaned gently with ethanol. The first and last measurement | a fresh filter paper, placed it in the detector and turned on the LED. We measured the resistance <i>R<sub>LDR</sub></i> directly with a multimeter. After each measurement the detector was cleaned gently with ethanol. The first and last measurement | ||
− | of each series | + | of each series were conducted with plain water to determine <i>R<sub>b</sub></i> and to confirm the absence of contaminations. A plot of the normalized resistances is shown in the figure below. We fitted the data with <a id="equation16">equation 16</a> to determine a value for <i>k</i> using a value of 0.8 for <i>γ</i> gives |
</p> | </p> | ||
<div class="equationDiv"><img class="largeEquation" src="https://static.igem.org/mediawiki/2017/d/d4/T--Munich--Hardware_equation17.png"><span>(17)</span></div> | <div class="equationDiv"><img class="largeEquation" src="https://static.igem.org/mediawiki/2017/d/d4/T--Munich--Hardware_equation17.png"><span>(17)</span></div> | ||
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</p> | </p> | ||
<p> | <p> | ||
− | With these quantitative data it is now an easy task to verify if the collateral RNase-activity of Cas13a got activated by | + | With these quantitative data it is now an easy task to verify if the collateral RNase-activity of Cas13a got activated by the binding of target RNA. For the convenience of the end user we choose to extract two kinds of information from the time traces produced with our detector. |
− | We check if | + | We check if an enzymatic reaction has taken place on the filter paper by evaluating whether the first 5 data points show a monotonous rise. Additionally we check if a threshold of 3 µM equivalent fluorescein concentration was crossed by looking at the last 3 data points. |
− | + | These two informations are computed in the following way: | |
− | if the reaction has taken place and the threshold was crossed the detector software will | + | if the reaction has taken place and the threshold was crossed, the detector software will show a plus, meaning "Pathogen detected"; if the reaction has not taken place but the threshold was crossed, the detector software will show a minus, meaning "Pathogen not detected". |
− | The other two combinations will lead to the | + | The other two combinations will also lead to the minus. |
− | The software will also create a graph similar to the graph in the beginning of this documentation to provide a deeper insight for the | + | The software will also create a graph similar to the graph in the beginning of this documentation to provide a deeper insight for the end user if needed. The software was written in Java, using the library for interfacing with the Arduino micro-controller. Right now it is optimized for Windows, but we plan on expanding it to more platforms. The code can be found on <a class="myLink" href="https://github.com/igemsoftware2017/igem_munich_2017">our github page.</a> |
</p> | </p> | ||
<p> | <p> | ||
− | We think that our detector is a more reliable read out system compared to other possibilities because | + | We think that our detector is a more reliable read out system compared to other possibilities, because Lightbringer provides deep insight into the reaction dynamics. Hence with an exact knowledge of Cas13a's reaction kinetics and further analytic tools, a nearly fail-safe read out can be achieved. |
− | + | A colorimetric readout, a binary change of color, in contrast, is way more primitive and provides no insight into any dynamics. | |
− | A colorimetric readout, a change | + | |
</p> | </p> | ||
</td> | </td> | ||
</tr> | </tr> | ||
+ | <tr><td colspan=6 align=center valign=center> | ||
+ | <h3>References</h3> | ||
+ | <p> | ||
+ | <ol style="text-align: left"> | ||
+ | <li id="ref_1">Wu, Jing, et al. "An economical fluorescence detector for lab-on-a-chip devices with a light emitting photodiode and a low-cost avalanche photodiode." Analyst 137.2 (2012): 519-525.</li> | ||
+ | <li id="ref_2">Walczak, Rafał, and Krzysztof Adamski. "Inkjet 3D printing of microfluidic structures—on the selection of the printer towards printing your own microfluidic chips." Journal of Micromechanics and Microengineering 25.8 (2015): 085013.</li> | ||
+ | <li id="ref_3">Yang, Feng-Bo, et al. "A low-cost light-emitting diode induced fluorescence detector for capillary electrophoresis based on an orthogonal optical arrangement." Talanta 78.3 (2009): 1155-1158.0039-9140, https://doi.org/10.1016/j.talanta.2009.01.033.</li> | ||
+ | <li id="ref_4">Novak, Lukas, et al. "An integrated fluorescence detection system for lab-on-a-chip applications." Lab on a Chip 7.1 (2007): 27-29.</li> | ||
+ | <li id="ref_5">Pais, Andrea, et al. "High-sensitivity, disposable lab-on-a-chip with thin-film organic electronics for fluorescence detection." Lab on a Chip 8.5 (2008): 794-800.</li> | ||
+ | |||
+ | </ol> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
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