iGEM Bielefeld 2015

Prototype Design

An easy way of detecting fluorescence

The problem

We planed to design a practical test strip using fluorescence as output signal. Green fluorescent protein (GFP) is among the most frequently used reporter proteins and has been used in numerous iGEM projects. But detecting fluorescence outside the lab sounded impractical to us. 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.

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.

In the beginning of our project, we wanted to test if it could work to photograph fluorescence with a smartphone. We got a filter from light engineering from the women cultural center Bielefeld e.V. ("Frauenkulturzentrum Bielefeld e.V.") and put it in front of the smartphone. In the picture below, we took a picture with these filter from purified GFP, sfGFP lysate and lysate without sfGFP. You can see different colors and a different brightnesses in the picture. So in our first impression we thought it's worth to try some other filters, to find the perfect filter for sfGFP imaging with almost no background signal.

The first filter test to get an rough impression if it could work to use two filters for 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 not induced sfGFP culture.

Therefore we bought a lee color filter catalog and began to test different filter combinations. The preselection was possible, because we had access to the light transmitting spectra of almost every filter.

The next step was to create a dark environment. Therefore we did handicrafts to design a "black box". Now it was possible to take photos under defined conditions and constant dark background.

The black box: tinkered to take photos under same conditions

The filter combinations

The essential requirements for taking comparable photos are done. Now we had to find the ideal filter combination. So we made a lot of photos with the preselection. We tested 6 filters for emission and combined every filter for emission with up to 16 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 filter combinations. On the top there is the picture without any filters. In the middle the picture was taken with fluorescence green in front of the camera and deep blue in front of the flash. The bottom photo was taken with the optimal filter combinition. Tokyo blue in front of the flash and twickenham green in front of the camera

As you can see in the picture 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 it out, we tried to photograph monomeric red fluorescent protein (mRFP) lysate, as well.

Test of different filtercombinations to photograph mRFP. On the top the picture is taken without any filters. In the middle the picture 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 filtercombinition. Twickenham green in front of the flash and light red in front of the camera.

Does fluorescence photography work on Paper?

So far so good. Fluorescence imaging of liquids in quite high amounts is possible. But does it also works with small volumes and on paper? Therefore 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 we got from Merck) and put the lysates on the paper and took the photos. Exemplary the results from laboratory filter paper from Merck are shown. The other ones showed the same results. As you can see in the picture below this paragraph, it is possible to photograph volumes of 5 µl purified GFP up to a concentration of 17,5 mmol/l.

We added a small amount of lysate to the scrap of paper and photographed it with the different filter combinations. The top photo is whithout any filter. The picture in the middle was taken with the filter combination for sfGFP and for the bottom picture the mRFP filters were used.

Now we wanted to find out if it is possible to photograph the fluorescence from a CFPS experiment on paper. So after a CFPS run on paper, we took the scrap of paper and took a photo from it. As you can see in the picture below.

We took the scrap of paper from the CFPS an took an photo. And as you can see in the picture it worked. We can take an photo of the sfGFP generated in the CFPS.

The App

Now it's possible to photograph fluorescence, but sometimes it's difficult to see the differences. And analyzing the pictures with the image processing software Fiji requires know-how of the user. On the one hand for the program and on the other hand for interpreting the results. We solved this problem with a smartphone App. 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%), valid biosensor signal is detected. Additionally it is checked whether the value for the positive control (drop of sfGFP) is above a certain value, thereby confirming the functionality of the biosensor. Furthermore the App displays specific information regarding the different heavymetals and date rape drugs to inform the user. For easy handling a correct positioning of the smartphone and the biosensor test strip, we designed and printed a measurement case.

A photo of our test strip generated by a typical smartphone 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 the pixels chosen are correct. Furthermore, it provides the values it calculated for the greeness of the spot.

The application is adapted to the box shown in the picture. The box provides the ideal environment for capturing pictures under reproducible conditions. The application will calculate whether a contamination is present and give out an list of the contaminants in the water sample.

The 3D print

Ok, the fluorescence detections works fine, but it's not really practical to take the photo. The filter have to be in the right position direct in front of the camera respectively of the flash. Also it has to be quite dark for high quality pictures. Therefore we designed a box and realized it with a 3D printer. The top can be changed specific for the smartphone you use. The test stripe can be placed on the push loading drawer and inserted into the box. So it's quite easy to use. You just have to put the smartphone on the top, insert the test strip an take the photo.

Team:Bielefeld-CeBiTec/Design