What is the common point between coffee, tea, Coca Cola and other energising drinks? Caffeine of course!
Caffeine is known to stimulate the central nervous system, a part of the nervous system including brain and spinal cord. That is why you could noticed that your heart rate was increasing after drinking a cup of coffee or any of the previous drinks. But is it the same for other species? During this Biosensor week about chemical gradients, we wanted to focus our study on Daphnias, small planktonic crustaceans of maximum 5 mm in length. It is an interesting organism for biologists as we can easily observe several organs thanks to its transparent body. So we decided to observe Daphnia’s heart: will it beat faster when the caffeine concentration of their environment is raised? The main goal of our project was indeed to find a correlation between caffeine concentration and their heart-rate, to finally use Daphnia as a biosensor able to determine the caffeine concentration of a solution. We chose to compare Daphnias with another sensor, a UV spectrophotometer. This tool quantify “how much does a solution absorb light?” as the optical density of the solution. So we expected that the more the solution is concentrated in caffeine, the more it absorbs light and therefore, the higher the optical density is.
We mentioned the term chemical gradient above, but you may ask, what is a chemical gradient? In biological systems, concentration imbalance is very important as different kinds of chemical substances are often unequally distributed on both sides of cells membranes for example. Hence, particles move from the higher concentrated region to lower concentrated region, activating lots of complex metabolic process.
In our case, we wanted to see how Daphnias and spectrophotometer would respond to different concentrations of caffeine, namely:
- No caffeine (0 g/L)
- 0,2 g/ L
- 3 g / L
How did we observe Daphnia’s heart reaction?
Figure 1: Experimental set-up
First of all we had to remove Daphnias from the main aquarium into a specific microscope slide with 10 wells (figure 2, figure 1: Sampling). Indeed, it allowed us to observe Daphnia individually and to prevent them from moving to much.
Figure 2 : A microscope slide with 10 wells and Daphnias inside
Once they were on the slide, we added one of the solutions thanks to a pipette (figure 1: Incubation).
We let Daphnia 5 minutes in this environment with caffeine. Indeed, we knew from a preliminary small study that the action of caffeine on Daphnia organism would be at its maximum after 5 minutes.
Finally, we could observe Daphnias with a binocular magnifier calibrated at the 5x magnification (figure 1: Observation). We counted the number of heartbeats of every Daphnias for 10 seconds. Are you curious about seeing a Daphnia’s heart closer? Look at this video !
Hint: Look at this drawing to localise their heart.
We studied 30 Daphnias for each concentration: with a large number of replicates we can reduce the noise in our measurements. Noise can be due to errors while analysing some Daphnias or to abnormal heart rate because of differences in Daphnia’s age, size or physical activity… So we used for the biological experiment more or less 90 Daphnias.
Figure 3: Results
Our results (figure 3) suggest that as expected, Daphnia’s heart rate effectively increased as the caffeine concentration was raised: in average, we counted 20 heartbeats more per minutes, which correspond to an increase of around 10%. However, Daphnias did not sense the difference between 0.2 g/L and 3 g/L concentrations, maybe because we had already reached the limit viable heart rate at 0.2 g/L of caffeine. Moreover, the 3g/L concentration killed the majority of Daphnias…
As we said before, we wanted to compare the sensibility of Daphnias to caffeine with the sensibility of an electronic tool: a spectrophotometer (figure 4).
Figure 4: The very spectrophotometer
We expected to find a proportional relationship between the concentration of a caffeine solution and its optical density, according to the very famous Beer-Lambert law in chemistry. To do so, we diluted a solution of caffeine several times by 10 to obtain 7 solutions from 1g/l to 10-6g/l. Then, we measured the optical density of each solution and… faced a problem. We obtain 0% for every solutions. We think that we should have diluted caffeine with sulphuric acid or used extracted caffeine rather than pure one.
Thus, in the context of our experiment, Daphnia is a better sensor of caffeine concentration than the electronic one, as their heartbeats were effectively affected by caffeine. To go further, we would like to test more concentration around 0.2g/L on Daphnias. The temperature and light exposure, which affect Daphnia’s heart rate, would better be controlled to decrease the experimental noise… Also we could test some method to make accurate heart rate count, using a stroboscope, usually used to make a cyclically moving object appear to be slow-moving, as it is explained in this article (Rachel Foster (1997) A stroboscopic method to investigate the effect of caffeine on Daphnia heart rate, Journal of Biological Education, 31:4, 253-255, DOI: 10.1080/00219266.1997.9655573): We would also improve the electronic sensor, or test another one (a pHmeter for example).
In any case, 1684 humans all around the world are sensing caffeine effects each second while drinking a cup of coffee...
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