Chronology of the prototypes
Here is a chronology of our different prototypes for the measurements of bioreporter eGFP fluorescence and turbidity :
Prototype 1 (2013)
The principe used is measuring the fluorescence at an angle of 90° from the light source.
The light from the LED passes through a lense to be focused and then through a filter, to only let the excitation wavelength pass through. That filtered light will excite the sample, which will emit at another wavelength and will then be detected. In general, photodiodes or photomultiplier are used as detectors, but they are quite expensive. That is why a digital camera is used as detector. Indeed, all camera contains an RGB filter, which can be used to detect only the range of wavelength of interest. A LED that emits at the needed range of excitation wavelength is used , to eliminate the presence of a filter.
For this device : two paperboard boxes sticked together with two holes for the camera and the LED are used. The box is closed so no external light would disturb the measurement. Inside, there is a horizontal place for the sample-holder and ouside we used an Arduino as the LED current source.
After testing, the device needs some improvements. For example, a lot of the components are outside of the box (like the LED and the camera), which makes it very difficult to take a fixed picture, because everything moves a little bit at each measure. The sample is positionned horizontally, which we discovered, is not the best orientation, because of light scattering, but also because we have to open the box for each sample. This is another factor that disturbs the system by making it move. Then we need a computer for the alimentation of the Arduino (that only acts as a battery).
So the improvements made :
- The addition of a battery to replace the Arduino and a switch, to be able to turn the LED on and off individually
- The fixation of the camera inside the device to obtain more precise pictures
- Putting the sample in a vertical position to allow an easiest change between different samples
Prototype 2 (2013)
To quantify the fluorescence of a sample, it is enough to excite it with the specific absorption wavelength in a dark room, filter the light emitted by the excited sample and then quantify it.
The idea is to use a LED generating light around 488 [nm] which is the eGFP absorption wavelength. The excited eGFP then emits light around 509 [nm]. To assure that the only light which is then quantified is the eGFP one, a filter which stops the 488 [nm] wavelength but let the 509 [nm] wavelength pass is inserted between the sample and the light quantifier.
This principle is shown in this scheme : The device works with an excitatory LED which light at 488[nm] excits the sample's eGFP, which light is filtered and quantified with the photoresistance
A lens could be necessary if the light is not enough intense to be quantified by the light quantifier. It only serves to concentrate the light in one point and so on the light quantifier device.
Another way to do it is to use two filters. One for the light filtration and another dichroic filter. As in the following figure, the dichroic is able to reflects certain wavelength and let pass others. So the idea is to reflects at 45° the excitation wavelength and then let the emitted light pass, filter a second time and then quantify the light.
The light quantifier could be a simple photo-resistance. The system we used was very simple: a photo-resistance was used as the R1 place of a voltage divider so that we can measure the voltage Vout through the arduino's microcontroller’s analogic pin. The photo-resistance, as its name indicates, is a simple electric resistance which resistance change proportionally to the light it receives. More the light increases, more the resistance decreases and more Vout tends to equal Vin. Inversely, more the light decreases, more the resistance increases and more Vout tends to be null.
The first version is build with a photo-resistance.
Cable connexions :
But, the device prototype build like this wasn't able to differentiate samples with different arsenic concentration because the photo-resistance was not enough sensitive to detect the light emitted by the dextran samples whereas they emit more light than the transformed bacteria. So we tried to change the light sensor and use the light to frequency component TSL235 in the version 2.1.
The device has been tested with different concentration of the fluorescent molecule dextran. The concentration was always diluted by two and the device always displayed a value which was the half of the previous value. So with the dextran's high fluorescence, we get the first results which confirm that this kind of device could work and which could distinguish different fluorescent intensities.
Another difficulty met, was to find the different components which prices aren't accessible to everyone (a filter can easy be about 300 CHF or more).
Prototype 3 (2014)
Our prototype is based on a fluorescent method where we use GMO bacteria expressing GFP (Green Fluorescent Protein) in presence of Arsenic in a water sample. This fluorescence is detected optically and can be quantified in order to measure the concentration of Arsenic.
A vial containing the water sample we want to test is positioned on a socket through which a blue LED passes. GFP absorbs this blue light (λ=475 nm) and emits green light (λ=504 nm) which is detected by a photosensor on which the light is concentrated with the help of two lenses that avoid loss of intensity and that allow more precise results.
Moreover, buildling on the version 2.0 design concept, a red LED was added to measure the turbidity. Turbidity is the cloudiness or haziness of a fluid caused by individual particles. In our case, if there are a lot of GFP, one GFP can absorb light emitted previously by another GFP and then emit light itself and thus creating a chain reaction falsifying the results. The measurement of turbidity will allow us to normalize our results.
We also added a screen interface for an easier reading of the results in the field.
With these measurements, one can determine the concentration of arsenic in the sample and so know if the water is drinkable or not.
The new features of the prototype v3.2 include :
- A red LED to account for scattering has been added next to the blue LED.
- All of the electronic components have been added to PCB’s and can be easily unsoldered and changed
- The position of the excitation and scattering LED underneath the sample have been set so that the vial will be completely illuminated, independently to the illumination angle of the LED’s (minimum = 20 degrees)
- The circuit has been adapted to control 3 LED’s and can supply a current of more than 1 A to each of them if needed.