Team:Moscow/Hardware
Hardware
To measure the fluorophore concentration (in our case, it is sfGFP by the emitted light, we need a specialized tool – a fluorimeter. There is a wide variety of commercially available options, but the high cost is one of the biggest limitations in student studies with a low budget. That's why we present a simple, sensitive, low-cost, and portable version of the fluorometer designed by our team. The prototype consists of a conventional 5mm LED, which is needed to excite the fluorophore (the excitation peak of sfGFP is at 485 nm, ice blue) two sheet filters — one between the light source and the sample cell, and the other between the cell and the detector, which is the camera module. All of the electronic components are connected to a microcontroller Arduino Nano.
Design
The prototype is a small box of 12x12x6 cm in size made of black PLA (polylactide) printed on a 3D printer. It consists of three parts — the main body, the lid, and the cap for the cuvette. The case is divided into several compartments: in the largest one, there is an Arduino Nano microcontroller with connections to the camera, button, and LED. It also has holes for these components to other compartments, as well as for a USB wire to connect it to a computer, laptop, etc. In the smallest compartment, there is a LED that goes out to a round window in the compartment with a cuvette. There is a small platform for attaching the cuvette. The round window is at a right angle to a square window, which leads to a long compartment with the camera module on the other end. The windows were designed for the filters so that they can be easily replaced. There are also holes in the corners where the lid legs are inserted. The lid itself on the underside has protrusions outlining the walls of the box for better opacity; the legs for attaching the lid, as well as the hole through which the sample cuvette is placed inside. Finally, to prevent the light from getting from above, we've additionally printed the cap for the cuvette. The prototype is made of black PLA for better light absorption. The prototype was made in collaboration with EPFL in Fusion360 according to the drawings of our team.
Components
Filters
One of the cheapest and most compact options was to use ROSCO filters, which we have found in the music store catalog. We had to choose from those filters that were available . R74 Night Blue (excitation filter) and E101 Yellow (emission filter) proved to be the most suitable ones, allowing to cut off unnecessary spectra and having a relatively high output efficiency (for example, R74 Night Blue has the transmission equals to 38% at 480 nm and E101 – 50-60% at 510 nm).
Detector
We have decided to use the OV7670 camera module for Arduino with a resolution of 640x480 and an overall size of 34x34 cm as a detector. It allows taking measurements in real-time and in low light, with a photographic frequency of up to 30 frames per second, and changing the parameters of the output image and the focus as well. And of course, it is much cheaper compared to a smartphone, whose photographic frequency and other characteristics vary from model to model. But there are also a couple of significant disadvantages — a huge number of connections that occupy almost all the pins, and complex programming of the device.
Measurement
To determine the fluorescence intensity – and hence the concentration of the fluorophore — one can use images obtained from the camera. We didn't have the opportunity to develop our own software that would allow us to determine the fluorescence intensity by images, so we had to look for other teams' decisions. Last year iGEM team Lambert created software based on the analysis of HSV images (hue, saturation, value) when average values are calculated from a given image and the value, which corresponds to brightness, is translated into arbitrary units. It helps to estimate the total value of fluorescence.