Team:Dalhousie Halifax NS/Model

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Fluorescence Model

Introduction

We were interested in building a model illustrating how pyoverdine fluorescence shifts given concentrations of aluminum (Al) and iron (Fe). The data used to build a model of pyoverdine fluorescence was taken from an article published in the Journal of Inorganic Biochemistry titled “Fluorescent complex of pyoverdin with aluminum”. Fluorescence intensities correspond to the sum of the emission values of pyoverdine between 450 and 470 nm [1]. The fluorescence emissions of different solutions of pyoverdine were measured with increasing ionic aluminum concentration and converted to intensity. In “Fluorescent complex of pyoverdin with aluminum”, the different pyoverdine solutions consisted of a 1 μM pyoverdine solution, a 1 μM pyoverdine solution with 3 μM Fe (III) added after aluminum, and a 1 μM pyoverdine solution with 3 μM Fe (III) and aluminum added simultaneously [1].

Fig 1. Fluorescence intensities of pyoverdine and Fe (III) solutions with increasing aluminum concentrations. Taken from figure 3 in “Fluorescent complex of pyoverdin with aluminum” [1].

The fluorescence intensity of 1 μM pyoverdine in the absence of metals is 2245. When pyoverdine is present in a 3 μM solution of Fe (III) without any aluminum in solution the fluorescence intensity is 80 [1].

Modelling

To build a model of pyoverdine fluorescence intensity with changing aluminum and iron concentrations we started with a basic assumption that the difference in fluorescence in solutions containing Al (III) only, and both Al (III) and Fe (III) is proportional to [Fe]. Using this assumption, we generated estimated values with different concentrations of Fe (III). These estimated values were converted to a ratio of fluorescence intensity by comparing them to the baseline fluorescence intensity of 2245, which was pyoverdine with no metals. Negative predicted ratios were set to 0.

Using the fluorescence intensity estimated data, and the pyoverdine and aluminum only intensity data from del Olmo et. al, a model of fluorescence intensity ratio was fitted to the following equation: y = b + a + f + f ² + a*f

Where y is the ratio of observed to baseline fluorescence intensity, a is the concentration of Al (III), f is the concentration of Fe (III), a*f refers to an interaction between a and f, and b is the fitted intercept. Simply put, this equation means that the ratio of observed to baseline fluorescence intensity depends on Al (III) concentration, Fe (III) concentration, Fe (III) concentration squared, and the interaction between the concentrations of Al (III) and Fe (III). Once the model was created Al (III) and Fe (III) concentrations from 0-4 were used as input as increments of 0.01 to develop a heat map using the above equation.

Fig 2. Fluorescence intensity ratio of observed intensity compared to the baseline intensity over varying Al (III) and Fe(III) concentrations.

As shown in Figure 2 an intensity ratio above 4 is only seen when there is a high concentration of Al (III), while having a low concentration of Fe (III). This model clearly demonstrates that there is no way to distinguish between cases where there is high Al (III) concentration and high Fe (III) concentration from cases when there is low Al (III) concentration and high Fe (III) concentration. The ratios for both of these cases are approximately zero. If the Fe (III) stayed at a low concentration, increasing the Al (III) levels over the above range will increase the fluorescence ratio from approximately 0 to 6.

Conclusion

Our model shows that only when iron is at low concentrations can differences between low and high ionic aluminum levels be detected. To implement this model into the use of our biosensor, determining the limit of the shift in fluorescence that our device can detect would tell us the range of ionic aluminum levels that can be distinguished. An important question to consider when developing the device is: given an output fluorescence ratio, how accurately can we say how much Al (III) and Fe (III) is there? One way to increase the accuracy of the model is to conduct future experiments determining the range of values that create a high intensity ratio. Incorporating an iron chelator will be important for our device since the model cannot distinguish between cases with high concentrations of both Al (III) and Fe (III), and with low Al (III) but high Fe (III) concentrations.

References

[1] del Olmo, A., Caramelo, C., & SanJose, C. (2003). Fluorescent complex of pyoverdin with aluminum. Journal Of Inorganic Biochemistry, 97(4), 384-387. doi: 10.1016/s0162-0134(03)00316-7