Future Directions

Due to the results we obtained, and the work we made during the semester, we have many ideas of which future directions could be taken, by us or by the next set of students active for this project.

Future directions are classified in two categories: improvements of our prototypes and general reflexions about the bacteria or the device that measures fluorescence.

Our prototypes:

• We need to do more experiments with arsenic and bioreporter. First, we need to know to what range of fluorescence our device can measure something. But we also need to know how the bacteria fluoresce and how its concentration distort the signal. Finally, we have to understand the function that relates arsenic concentration to the datas finally processed with our prototypes. During this semester, we analyzed a bit these different questions,but always separately one from another. It means that we have no ideas how the different aspects relate one with another... and the only integrated experiment we did gave no interpretable results as we were not prepared enough and had not sufficient knowledge about that.
• We have to test our LEDs. We have to be sure that we have the right excitation wavelength. Imagine our LEDs are sligthly lighting with a wavelength that pass the optical filter: in that case, the results would be distorted a lot because the enery of the LEDs is far greater than the energy of the fluorescence due to GFP. Furthermore, we have to test which wavelenght is preferred by our fluorescent protein and which wavelength range is the best to both excitate and evitate a contamination of the signal.
• For the second prototype, we have to test filters. As for the LEDs, it is important to eliminate any risk of pollution of the signal by examining if the filters are correctly filtering and to what range. If we cannot eliminate all contamination by light, we have to know the range it occupies in order to function with it.
• For the second prototype, we have to add lenses. The fact is that the light from the fluorescing sample is passing the filters only if the angle with the filter is lower than a certain value. Hence, we are loosing a big part of the signal, and as the signal is tiny, it is a pity. With a lens, we could have more light passing the filter and a better activation of our photoreceptor.
• For the second prototype, we can improve it in order to get rid of the computer: we could have an integer circuit and find a way for the results to be displayed on a screen attached to the circuit.
• For the first prototype, we could improve CHDK use in order to analyze directly the picture in the camera. Such an analysis would allow a faster result and a cheaper and more portable device, because no computer would be needed anymore.
• We could design the first prototype to be used with a smartphone. Indeed, a big part of the population nowadays own a smartphone, so it would respect the accessibility criteria. Furthermore, an application ImageJ (the programm we use for analysis) exists for smartphone. We could also create a whole application, taking the picture and directly measuring the fluorescence intensity.
• If we continue using a picture and ImageJ for the first prototype, we can find a way to improve reception, making it closest to 509nm instead of the range of 497nm to 560nm that serves now for the reception of the signal.
• Finally, we could improve the first prototype to have many samples pictured at the same time. This would be a possibility to compare directly samples between them or compare to a blank value, or a known Arsenic concentration.

General future reflexions:

• There is a big reflexion that need to be made about the sample holder. The sample-holder must cumulate a wide range of properties: confinement, growth environment for bacteria, possibility for the light to enter, excitate and being emitted and collected. We could imagine a bit of everything, such as PDMS microfluidics holder, test-tube, sealed tube, plate, sealed plate... We have seen mostly two possibilities: a microfluidics sample-holder, and a bigger, stationnary sample-holder. Microfuidics brings many advantage for the waste size, the movement to which bacteria are submitted, the signal accessibility. But it also brings difficulties in the creation of a system able to measure fluorescence, and in the precisness needed for the instruments used. A bigger sample-holder as we used does not provide a nice growth environment if we do not move it while bacteria are processing the Arsenic signal. We also have to think to the Oxygen supply during the growth (leaving an empty space) without disturb the measures of the light with bubbles. With a big sample-holder we also have to know the distance the signal is traveling before being measred: it is possible that the signal is lost by diffraction in a to big volume. But a big sample-holder is simplier to manipulate and less expensive to produce. Hence, the sample-holder is a nice piece for reflexion and we hope a best solution than the one we use actually will be found.
• One of our biggest problem in the analysis of fluorescence with the bateria we use is the fact that it is expressing eGFP and that eGFP needs an excitation wavelength that is very near the emission wavelength. This proximity makes the filter design difficult. One possibility would be to engineer the bacteria such that the fluorescent protein expressed in presence of Arsenic reacts with different wavelength. Changing the fluorescent protein would also have a cost advantage. Indeed, finding a LED lighting in UV range is far more difficult and expensive than one in the visible spectrum or in infrarred range. Having a fluorescent protein reacting to excitation at a longer wavelength would be interseting in terms of cost an accessibility of the prototype.
• With GFP expressing bacteria, the analysis of Arsenic concentration in water require nearly three hours. this is already faster than going in a lab and waiting days before having a result. But this delay could also be ameliorated if we tried to use another reporter. This is a project that could last years! One idea could be to use luciferase.
• During the semester, we discovered that working with bacteria is not that simply reproducible. The detection of Arsenic is dependent of the number of bacteria, their ability to react, the phase they are in as much as the concentration of Arsenic itself. Assuming we want to end in a reproducible device measuring Arsenic, we would need to define a function that would allow us to infer Arsenic concentration from the fluorescence quantity. This function would be dependant of the number of bacteria, their survival rate, the temperature, the oxygen and other nutrients available in the sample, the movement and other forces they are submitted to... Finding this function need a long time experimentating and a very big number of data. Otherwise, when measuring, we would always need to have samples used as negative and positive controls, such as a sample with a known concentration of Arsenic, in order to compare to the measured sample and guess its Arsenic concenration. So working with bacteria is not that simple and we could also continue researches to find a more reproducible way to measure Arsenic... which is a new adventure!