Difference between revisions of "Team:Bielefeld-CeBiTec/Design"
| Line 168: | Line 168: | ||
| − | <div class="The | + | <div class="The app"> |
<div id="app"> | <div id="app"> | ||
<h2> The App </h2> | <h2> The App </h2> | ||
| − | <p>Now it's possible to photograph fluorescence, but sometimes it's difficult to see the differences. | + | <p>Now it's possible to photograph fluorescence, but sometimes it's difficult to see the differences between the divers paper discs. Additionally analyzing the pictures with the image processing software Fiji requires know-how of the user. For both the program and the interpretation. We solved this problem with a smartphone app. We coded the app with Android Studio 1.2.2 and can be installed on smartphones on Android 4.2 (Jelly bean) and later. The app determines the median of the greenvalue of a area of pixels in the sensorspot and compares it to the pixels of the negative control (the not induced sensor). If the ratio of a sample and the negative control is above a certain theshold (10%), a valid biosensor signal is detected. Additionally it is checked, if the value for the positive control (drop of sfGFP) is above a certain value, confirming the functionality of the biosensor. Furthermore the app displays specific information regarding the different heavymetals and date rape drugs to inform the user. You can download it by clicking on the button below. You need to unzip the file on your phone, since .apk files can not be uploaded. Alternatively download it <a href="https://2015.igem.org/File:CellFreeSticks.apk.txt">here</a> and delete the fileextension ".txt".</div> |
| − | + | We designed and printed a black case for an easy handling and a correct positioning of the smartphone and the biosensor test strip. | |
<br> | <br> | ||
</p></div> | </p></div> | ||
| Line 181: | Line 181: | ||
<figure><center> | <figure><center> | ||
<img src="https://static.igem.org/mediawiki/2015/b/b2/Bielefeld-CeBiTec_app1small.png"> | <img src="https://static.igem.org/mediawiki/2015/b/b2/Bielefeld-CeBiTec_app1small.png"> | ||
| − | <figcaption>A photo of our test strip generated by a typical smartphone using an ideal filter combination to detect fluorescence of sfGFP.</figcaption></center> | + | <figcaption>A photo of our test strip. The photo was generated by a typical smartphone (samsung galaxy s5 mini) using an ideal filter combination to detect fluorescence of sfGFP.</figcaption></center> |
</figure> | </figure> | ||
</div> | </div> | ||
| Line 187: | Line 187: | ||
<figure><center> | <figure><center> | ||
<img src="https://static.igem.org/mediawiki/2015/8/80/Bielefeld-CeBiTec_app2small.png"> | <img src="https://static.igem.org/mediawiki/2015/8/80/Bielefeld-CeBiTec_app2small.png"> | ||
| − | <figcaption>Screenshot of the app. It shows the pixels it took into account for the calculation in red, so the user can check, whether the pixels chosen are correct. Furthermore, it provides the values it calculated for the greeness of the spot.</figcaption></center> | + | <figcaption>Screenshot of the app. It shows the pixels it took into account for the calculation in red, so the user can check, whether or not the pixels chosen are correct. Furthermore, it provides the values it calculated for the greeness of the spot.</figcaption></center> |
</figure> | </figure> | ||
</div> | </div> | ||
| Line 194: | Line 194: | ||
<figure><center> | <figure><center> | ||
<img src="https://static.igem.org/mediawiki/2015/e/e0/Bielefeld-CeBiTec_app3small.png"> | <img src="https://static.igem.org/mediawiki/2015/e/e0/Bielefeld-CeBiTec_app3small.png"> | ||
| − | <figcaption> | + | <figcaption> Combination of the app and the black case. The black case provides the perfect environment for capturing pictures under reproducible conditions. The application will calculate whether a contamination is present and give out a list of contaminants in the water sample.</figcaption></center> |
</figure> | </figure> | ||
</div> | </div> | ||
| Line 212: | Line 212: | ||
<div class="The 3D print"> | <div class="The 3D print"> | ||
<h2> The 3D print </h2> | <h2> The 3D print </h2> | ||
| − | <p> | + | <p> We showed that, the fluorescence detection works fine, but it's not very practical to take the photo. The filters have to be in the exact right position directly in front of the camera and the flash. It also has to be quite dark for high quality pictures. Therefore we designed a black case and realized it with a 3D printer. Marco Radukic designed the case in exact accordance with our guidline. The top smartphone inlay can be specifically changed for different smartphone types. The test stripe can be placed on the push loading drawer and inserted into the box. Thus it is quite easy to use. The smartphone is put on the top inlay, the test strip is inserted and the taken photo provides information about the contamination. To download the model for the 3D printing click <a href "https://2015.igem.org/File:Bielefeld-CeBiTec_black_case_3D_Druck_Datei.zip"> here. </a></p> |
| + | <br> | ||
<figure style="margin:auto; width: 600px"> | <figure style="margin:auto; width: 600px"> | ||
| − | <a href="https://static.igem.org/mediawiki/2015/9/98/Bielefeld-CeBiTec_3D_box_modell.png" data-lightbox="detection" data-title="On the left side you can see the 3D modell of the case, realized by Macro Radukic in exact accordance with our guidline. On the right you can see the printed black case. Printed from Thomas Schäfer with his 3D printer "><img src="https://static.igem.org/mediawiki/2015/9/98/Bielefeld-CeBiTec_3D_box_modell.png" ></a> | + | <a href="https://static.igem.org/mediawiki/2015/9/98/Bielefeld-CeBiTec_3D_box_modell.png" data-lightbox="detection" data-title="On the left side you can see the 3D modell of the case, realized by Macro Radukic in exact accordance with our guidline. On the right you can see the printed black case. Printed from Thomas Schäfer with his 3D printer. "><img src="https://static.igem.org/mediawiki/2015/9/98/Bielefeld-CeBiTec_3D_box_modell.png" ></a> |
| − | <figcaption> On the left side you can see the 3D modell of the case, realized by Macro Radukic in exact accordance with our guidline. On the right you can see the printed black case. Printed from Thomas Schäfer with his 3D printer | + | <figcaption> On the left side you can see the 3D modell of the case, realized by Macro Radukic in exact accordance with our guidline. On the right you can see the printed black case. Printed from Thomas Schäfer with his 3D printer. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
Revision as of 20:53, 18 September 2015
Output Signal processing
A Prototyp for easy fluorescence detection
The problem
We planned to design a convenient test strip using fluorescence as an output signal. Green fluorescent protein (GFP) is one of the most frequently used reporter proteins and it has already been used in numerous iGEM projects. Detecting fluorescence outside the lab sounded impractical to us and so we had to find an easy way to analyze the output.
The first steps
Before testing we decided to measure the extinction and emission spectra from sfGFP as a basis to choose the most promising filter combination and to have a look if the smartphone flash really excites sfGFP.
In the beginning of our project, we tried to photograph fluorescence with a smartphone. We got a filter from light engineering by the women cultural center Bielefeld e.V. ("Frauenkulturzentrum Bielefeld e.V.") and put it in front of a smartphone. In the figure below, we took a picture with this filter from purified GFP, sfGFP lysate and lysate without sfGFP. You can see different colors and a different brightnesses in the picture. In our first impression we thought it's worth to try some other filters, to find the perfect filter or filter combination for sfGFP imaging with almost no background signal.
Therefore we bought a lee color filter catalog. We made a preselection of the filters. This was possible, because we had access to the light transmitting spectra of almost every filter. With this preselection we began to test the different filter combinations.
The next step was to create a dark environment. Therefore we did handicrafts to design a black carton. Now it was possible to take photos under defined conditions and with a constant dark background.
The filter combinations
The essential requirements for taking comparable photos are achieved. Now we had to find the ideal filter combination. We made a lot of photos with the preselected Filters. We tested every combinition of 5 filters for emission with 12 filters for extinction. After analyzing the photos with the image processing software Fiji, the optimal filter combination found was tokyo blue (071) in front of the flash and twickenham green (736) in front of the camera.
As you can see in the pictures above it is really important to choose the right filters to get a high fluorescence signal and a low background signal. But is it only possible to photograph sfGFP and GFP? To find out, we took photos of the monomeric red fluorescent protein (mRFP) lysate, as well. For this protein we determined Twickenham green (736) in front of the flash and light red (182) in front of the camera to act as the optimal filter combination.
Does fluorescence photography work on paper?
We showed that fluorescence imaging of liquids in quite high amounts is possible with our approach. But does it also work with small volumes and on paper? To assess this we took normal laboratory filter papers. (MN 827 B from Macherey and Nagel, C 350 L and FN3 from Munktell and a laboratory filter paper from Merck), put the lysates on the paper and took photos with our filter systems. Exemplary the results of the laboratory filter paper from Merck are shown in the figure below. The other filter papers showed the same results. As it can be seen see, it is possible to photograph volumes of 5 µl purified GFP up to a concentration of 17,5 mmol/L.
Now we wanted to find out if it is possible to photograph the fluorescence from a Cell-Free Protein Synthesis (CFPS) on Paper. After a CFPS run on paper, we took the paper discs and took the photo shown below.
The App
Now it's possible to photograph fluorescence, but sometimes it's difficult to see the differences between the divers paper discs. Additionally analyzing the pictures with the image processing software Fiji requires know-how of the user. For both the program and the interpretation. We solved this problem with a smartphone app. We coded the app with Android Studio 1.2.2 and can be installed on smartphones on Android 4.2 (Jelly bean) and later. The app determines the median of the greenvalue of a area of pixels in the sensorspot and compares it to the pixels of the negative control (the not induced sensor). If the ratio of a sample and the negative control is above a certain theshold (10%), a valid biosensor signal is detected. Additionally it is checked, if the value for the positive control (drop of sfGFP) is above a certain value, confirming the functionality of the biosensor. Furthermore the app displays specific information regarding the different heavymetals and date rape drugs to inform the user. You can download it by clicking on the button below. You need to unzip the file on your phone, since .apk files can not be uploaded. Alternatively download it here and delete the fileextension ".txt".
The 3D print
We showed that, the fluorescence detection works fine, but it's not very practical to take the photo. The filters have to be in the exact right position directly in front of the camera and the flash. It also has to be quite dark for high quality pictures. Therefore we designed a black case and realized it with a 3D printer. Marco Radukic designed the case in exact accordance with our guidline. The top smartphone inlay can be specifically changed for different smartphone types. The test stripe can be placed on the push loading drawer and inserted into the box. Thus it is quite easy to use. The smartphone is put on the top inlay, the test strip is inserted and the taken photo provides information about the contamination. To download the model for the 3D printing click here.
