The problem

We planed to design a practical test stripe using fluorescence as output signal.Green fluorescent protein (GFP) ist among the most used reporter protein and has been used in numerous iGEM projects. But fluorescence outside the lab sounds not really simple to use. So we had to find an easy way to analyze the output.

The first steps

We planed to design a practical test stripe using fluorescence as output signal. But fluorescence outside the lab sounds not really simple to use. So we had to find a easy way to analyze the output. But 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 extincts sfGFP.

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.

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 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.

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.

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.

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.

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.