Analyse the Results
Units and Beer’s law
The experimental team used a plate reader, which measured the changes of absorbance over time in optical density values. To convert these into concentrations of ABTS, for comparison with our model predictions, we used Beer’s law and a calibration experiment. This allows the experimental results to be directly compared with the model predictions and hence further analysis to be undertaken.
The calibration experiment was done with ABTS, H2O2, and HRP. There was an excess of H2O2, so that the endpoint of the reaction was such that we could assume all the ABTS was oxidised. We did this experiment for a range of ABTS concentrations. There were some extra complications because ABTS decays and the other reagents cause absorbance; these issues were taken into account initially, but the influence on the results was negligible so this assumption could be relaxed. This analysis allowed us to convert OD values to concentrations in μg ml-1 ABTS, using Beer’s law, which states that absorbance measured in OD values at a given path length is proportional to the concentration of the absorbing compound (dye). Using the known molecular weight of ABTS, this could then be converted to concentrations in mM, which could be compared to our model predictions.
How we did this for the experiments?
The experimental team used a plate reader. The units showing concentration over time were optical density values, these can be converted into a concentration using beers law and a calibration experiment.
Our OD-to-concentration converter software
The code for these calculations is available on our [Github page]. Absorbance values in OD are read into MATLAB, together with the concentrations of oxidised ABTS from the calibration experiment. A linear regression line is then fit to the data. Using the equation of the resulting calibration line, it is now possible to convert absorbance values (OD) to concentrations (μg ml-1.) Dividing the result by the molecular weight of the compound (g mol-1) results in the concentration (μmol ml-1 = mM).