Difference between revisions of "Team:Newcastle/Notebook/Lab/AlternativeConstructs"

The best fit for this data series is a power series ($R^{2}=0.9873$). This means that initially small changes in concentration cause a large change in the resistance until the solution becomes saturated and this starts having less of an effect. This is clearly shown on the below graph.

For us, this confirms two important things. Firstly, that we can adjust the conductivity/resistivity of our media easily by varying the amount of salt. For example, increasing the amount of salt in LB. Secondly, that only a small amount of salt is required to improve conductivity. This latter point is important because our bacteria are unable to survive at high salt concentrations due to osmotic pressure. Most E. coli strains can only tolerate up to 0.5 M NaCl.

The second medium we used was LB. We would expect this to follow a similar trend to the NaCl solutions as it is the salt components of LB that allow it to conduct electricity.

Indeed, we do so this trend, albeit with higher resistances due to the further dilution of the salts for 40-100% LB in water. It appears to us that the result for 20% LB is anomalous.

This gives us useful baseline resistances for estimating the best LB concentration to use to generate our desired heating effect. The downside to LB however, is that it is difficult to replicate the preparation each time, compared to a minimal media like M9. We had hoped to also test M9 media as part of this experiment but precipitates had formed in our preparation.

Our final media was gold nanoparticles in PBS. As with the salt solution, we were investigating whether we could finely control the resistance of the media using additives. As pointed out previously, our bacteria are not tolerant to salt and it is metabolically active which means we could be interfering with cellular processes by altering the concentration. This may have an adverse effect on protein production. Gold, by contrast, is not metabolically active so we can add it to the growth media without any effect on the cells.

We had hypothesized that adding the gold nano-particles would decrease the resistivity of the media, that is the gold nanoparticles would facilitate better conduction. The gold nanoparticles that we used are a 20 nm suspension in 0.1 mM PBS from Sigma-Aldrich.

Because the gold is suspended in PBS we used that as the dilution medium. We added gold nano-particles to 0.5x PBS at concentrations of 0-100% (e.g. 20% nanoparticle solution to 80% PBS). The data we collected is shown in the below table.

Our data runs contrary to what we expected, increasing the number of gold nanoparticles increases, the resistance of the solution, as you can see from the graph below.

Unfortunately, this means that we will be unable to use the gold to decrease the resistance, though we can use it to increase the resistance if required. We are more likely to have to decrease the resistance rather than increase it, since the power is proportional to $I^{2}R$ we can improve the power dissipated in the media (and therefore the heating effect) better by increasing the current rather than resistance. This is illustrated in the below graph which shows that the increase we gain from varying the resistance grows linearly whilst the varying the current allows the power to grow exponentially.

These values are linked, $I = \frac{V}{R}$, so there is a trade-off between increasing or decreasing the resistance. Further experiments are required to determine what the optimal resistance for best power output is. However, we now know that using salt and or gold nanoparticles we can vary the resistance in either direction which is a useful tool.

It occurred to us during this experiment that the gold may also affect the heating rate of the medium. We will conduct future experiments in this area to determine this effect.

Computing Required Voltage

We can use the data we have collected from this experiment to determine the voltage required to heat the media in our microfluidics chambers. We do this by linking the energy needed to heat water, given by $E = C_{w}\cdot \Delta T\cdot mass$, and the energy dissipated by a resistor. In the equation for the energy required to heat water $C_{w}$ is the specific heat capacity of water. This formula can be used for any liquid for which we know the specific heat capacity. Thanks to our collaboration with Exeter we know that LB media behaves similarly to water (since water makes up the majority of the media) so we can assume a similar specific heat capacity. We had performed bomb calorimetry experiments to determine the exact specific heat capacity of LB but these were unsuccessful. The specific heat capacity we use here is 4 J/Kg. From our empirical experiments with microfluidcs we found that a heating time of 3 minutes was best to prevent pressure build up from electrolysis. This means the required energy is $E = 4 \cdot 0.1 \cdot (3 \cdot 60) = 72W$ as our chamber holds 1 ml of fluid, weighing 0.1 Kg.

The equation for the energy dissipated is $E = Q \cdot U = I \cdot s \cdot U = \frac{U^{2} \cdot s}{R}$.

By relating the two, $C_{w}\cdot \Delta T\cdot mass = \frac{U^{2} \cdot s}{R}$ and solving with the average resistance we find that to get the desired heating effect in our microfluidics chambers we need a supply voltage of approximately 30 volts. This is the DC voltage, as we intend to use AC this is actually the root mean square value, and the peak voltage required is actually $V_{p} = V_{RMS} \cdot \sqrt{2} = 30 \cdot \sqrt{2} \approx 42V$.

19/08/2016 - Gold Nanoparticle Heating Experiment A

We wondered if the gold nanoparticles could be used to enhance the heating effect within the microfluidic chamber, and reduce the time taken for the requisite temperature change to induce the heat shock response.

We found little correlation between the results, and decided not to proceed further with this approach given our time constraints.

22/08/2016 - Rhodamine B Flourescence Experiment A

Following on from our Electrochemical Impedance Spectroscopy work we wanted to determine the heating effect of applying our computed voltage to LB media. Because the heating is being performed in microfluidics chambers it is difficult to measure directly using a thermometer. We found that the thermometer crushes the chamber causing liquid to leak from it when it is not inserted carefully. To overcome this issue we investigated using fluorescent dye as a temperature measure. In particular we chose the well used and characterized dye Rhodamine B (Karstens, T., 1980).

Rhodamine B has the emission spectra shown below with a peak emission at 565nm. It has the property that the fluorescence yield varies with temperature. It is this property which makes it useful for measuring temperature in situations where a thermometer or other technique would be too invasive, such as in biological samples (Chen & Wood, 2009).

Before using the dye in our experiments we first wanted to test that we could measure the fluorescence signal using the equipment we had available. To do this we proceeded as per Shah et al. (2009) which pertains specifically to microfluidics experiments and prepared a solution of 0.1 mmol/L Rhodamine B in LB broth.

We then heated our solution to 55 °C and allowed it to cool. At various temperatures shown in the dataset below, we took a 0.5 ml sample and measured the fluorescence using a plate reader. The data we collected is shown below.

We aborted the experiment because we did not appear to be getting correct data, there was no variation in fluorescence. We believed this could have been because the plate reader was set up incorrectly to measure the emission wavelength of the Rhodamine B and so postponed the remainder of this experiment until we could use the new plate reader.

02/09/16 - First Bulb Current Test

We breifly tested the DNA2.0 lightbulb construct (htpG) in our microfluidics chambers. We placed 1.25 ml of transformed E. coli liquid culture after 24 hours growth into two chambesr using the 'DNA2.0 1(a) 50 31/08/2016' sample.

We observed before we conducted the experiment that the cells were not glowing on exposure to a UV LED as we would expect. We then subjected one chamber to 12 mA current (DC) for 90 seconds and the other to 15 mA for 60 seconds. It was originally intended that we apply the current for longer as we found it takes 2-3 minutes for our desired temperature change in LB at these currents, however the chambers leaked through the PDMS-slide interface which caused the circuit to break early and the power supply to shut off.

To prevent this from happening in the future we think it would be best to adapt our current chamber designs into 3D printed ABS which would form one complete sealed unit so that no leak could occur.

We then recovered the liquid remaining in the chambers and plated them out to see if they grew overnight. Note that due to the leakage the recovering volume was very small (~0.5 ml).

References

1. Chen, Y.Y. and Wood, A.W., 2009. Application of a temperature dependent fluorescent dye (Rhodamine B) to the measurement of radiofrequency radiationâ induced temperature changes in biological samples. Bioelectromagnetics, 30(7), pp.583-590.
2. Karstens, T. and Kobs, K., 1980. Rhodamine B and rhodamine 101 as reference substances for fluorescence quantum yield measurements. The journal of physical chemistry, 84(14), pp.1871-1872.b
3. Shah, J.J., Gaitan, M. and Geist, J., 2009. Generalized temperature measurement equations for rhodamine B dye solution and its application to microfluidics. Analytical chemistry, 81(19), pp.8260-8263.