Fluorolion makes detecting fluorescence much easier
In our project, we want to detect antigens in very small concentrations with a self-amplifying system. The plan is to develop this system in such a way that a test can be carried out quickly and easily by the family doctor. Therefore the system of detecting the antigen has to be easy and quick. Our system's detection component is based on a FRET pair's fluorescence. However, fluorescence can currently only be measured with large, complicated instruments which are not available everywhere. We aim to make the fluorescence of our test easier to measure, also with a view to our system being used in a test format by a family doctor or even as a self-test. So we developed a box and printed it with a 3D printer. We optimized it in several cycles by looking at the use of the box from a user's perspective.
If you want to measure fluorescence, you must place your samples in the prefabricated compartment and slide them into the box. Then you place your mobile phone on the box and can see the fluorescence of the sample through its camera. This has the advantage that the detection of our FRET pair would also be quite easy in real value, without the need for large, expensive, and incomprehensible devices. With further optimization, this system could also be made smaller and even more usable.
How is the system used?
First, a Petri dish with the sample to be examined is placed in the drawer and then pushed into the box. By pressing the button, the lysates of the three components of our system are pumped into the Petri dish inside the box. This is done with the help of three pumps, which are located in the box and each contain one of the three lysates of our system. Now a photo can be taken with the help of the flash light function of a mobile phone. The result can now be seen in the picture. Yellow = no antigen present. Cyan = antigen present.
But how exactly does the fluorescence detection work?
Two filters are used to measure the fluorescence. One filter (filter 1) is held in front of the flashlight of the mobile phone and the other filter (filter 2) is held in front of the camera of the mobile phone. Filter 1 brings the light of the flashlight to the right wavelength to excite the mTurquise2, the donor fluorophore of the FRET pair. If this is then excited by the light, there are two possibilities: If the antigen is present, the protease has cut the linker between mTurquise2 and EYFp. This means that the distance between the two dyes is no longer possible for energy transfer via the FRET system. So the sample emits the cyan-coloured light of mTurqise, which is picked up by the second filter in front of the camera. If the antigen is not present, the linker between the two dyes still exists and the energy transfer can take place. After mTurqise2 has been excited by the filtered light of the flashlight, it transfers its energy to EYFP when the electrons fall back to the ground state. This excites EYFP and raises it to a higher energy level. When the electrons of EYFP fall back, yellow light is emitted, which becomes visible through the camera. Now the result can be read off.
In the two pictures you can see how fluorescence was recorded in the fluorolion. We put our FRET pair as a sample in a Falcon tube and in a microtiter plate in the box. Then we attached the two filters to the mobile phone as described above and placed the mobile phone on the box. Then we took a picture with flash and we could see the fluorescence of the sample on the picture
Future plans
In order to improve the measurement itself, but also to make the change of antigen concentrations, for example within a disease progression, more detectable, it would be nice to develop an app in the future. With the help of this app, it would then be possible to store measurement data, which would make it easier to depict the course of a disease. In addition, it would be possible to have the measurement data analysed and printed out, for example, in order to file them in the medical record.
To know more about how the box look like and which steps were necessary to develop the box, have a look at the pages Engineering success and Hardware.