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Fluorescent spectrophotometry

Background

*PLACE HOLDER TEXT - Fluorescent spectrophotometry is a technique used to quantitatively measure the fluorescence of a fluorophore at a specific wavelength (reference?). This is done by exciting the fluorophore at a specific wavelength, causing the light to be emitted back at the wavelength measured. This technique will eventually be used in our final product to be able to tell if there is a positive result for bovine tuberculosis (bTB). In our experiments we used this technique multiple times and therefore needed to create a measurement to allow our results to be more easily interpreted; this was achieved by designing concentration curves using the same settings and volume of samples that were used to measure our other experiments. These concentration curves could then convert arbitrary units (AU), which could only be interpreted using our spectrophotometer on the right settings, to fluorescein concentration equivalents (CFE) which can be used across all laboratories.

Why we did this

Fluorescein was chosen due to being available in the iGEM 'Fluorescence Measurement Calibrants Kit' therefore allowing future iGEM teams to easily understand our results, as well as being a relatively cheap fluorophore allowing many people to have access to it. Fluorescein was the prefect fluorophore to model a concentration curve around, this is due to the fluorophore in the RNase Alert probes also being fluorescein. This means that our model will have the same extinction coefficient (E=) (Reference) and a very similar fluorescence spectrum to what will be used in our tests, meaning the same excitation and emission wavelengths can be used for greater accuracy of the concentration curve.

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