Revision as of 21:25, 18 September 2015

iGEM Bielefeld 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.

Emission and extinction spectra of sfGFP. The characteristic curve from the samsung galaxy S5 mini flash is also shown. The overlap of the different spectra shows that it is possible to excite sfGFP with a smartphone flash.

First filter test. It was executed to get an indication wheather or not two filters are sufficient for a fluorescence imaging. We used one filter in front of the flash and the same one in front of the camera. In the picture you can see purified GFP, sfGFP lysate and the lysate of a non induced sfGFP culture.

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 black box: tinkered to take photos under same conditions

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.

Test of different filtercombinations. On the top there is the picture without any filters. In the middle the picture was taken with fluorescence green (219) in front of the camera and deep blue (120) in front of the flash. The bottom photo was taken with the optimal filter combinition of tokyo blue (71) in front of the flash and twickenham green (736) in front of the camera

Test of different filtercombinations to photograph mRFP. On the top the picture is taken without any filters. The picture in the middle was taken with light red in front of the camera and dark yellow green in front of the flash. The bottom photo was taken with the optimal filter combinition twickenham green in front of the flash and light red 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.

We added 5 µL of lysate to the paper disc and photographed it with the different filter combinations. In the first row dilution seriesof purified GFP were added to the paper discs. In the second row sfGFP lysate were added, in the third row mRFP lysate was used and in the last row lysat without sfGFP and mRFP were added to the paper discs. The top photo was taken without any filter. As you can see, there is no fluorescence visible. The picture in the middle was taken with the filter combination for sfGFP. In this picture you can see two glowing paper discs in the first and in the second row. In the bottom picture the mRFP filters were used. Three paper discs of the third row are red. So it's possible to detect the mRFP lysate up to a 1:10 dilution.

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.

Photo of the CFPS run on paper. We took the paper discs of paper from the experiment on paper, took a photo and it worked, hence we can take a photo of the sfGFP generated in a CFPS experiment. In the first row you can see purified GFP, a dilution of the GFP and sfGFP lysat. We used this row for comparison. In the second row is the negativ control. We need the negativ control to visualize the self-fluorescence of the cell extract. In the third row are the paper discs where our self made extract were added for the CFPS. In the last row you can see the paper disc with the commercial cell extract for CFPS.

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".

We designed and printed a black case for an easy handling and a correct positioning of the smartphone and the biosensor test strip.

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.

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.

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.

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.

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.

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-<p> Now we wanted to find out if it is possible to photograph the fluorescence from a <a href="#paperCFPS">Cell-Free Protein Synthesis (CFPS) on Paper</a>. After a CFPS run on paper, we took the paper discs and took the photo shown below. </p>
+<p> Now we wanted to find out if it is possible to photograph the fluorescence from a <a href=""#paperCFPS" target="_blank">Cell-Free Protein Synthesis (CFPS) on Paper</a>. After a CFPS run on paper, we took the paper discs and took the photo shown below. </p>
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