The degradation rate of explosive compounds in-vivo using Raman spectroscopy
Introduction
The aim of this experiment will be to measure the degradation rate of explosive compounds in-vivo using Raman spectroscopy. The degradation rate of enzymes Nem-A and Xen-B when transcribed by an E. coli bacterium will be measured. In addition to the degradation rates a value for the partition coefficient for the explosive compounds between water and the cell membrane will be determined.
Raman Spectroscopy
Raman spectroscopy is a technique used to observe vibrational, rotational and other low frequency modes in a system. It utilizes the inelastic scattering of monochromatic light from these low frequency modes by detecting the shift in reflected photon energy. This shift can be used to give information about the types of bonds in the system and how many of these are present.
Typically the inelastically reflected light from the low frequency modes in a system is very weak in comparison to the elastically scattered light from the sample and in the past this had been the major barrier to Raman spectroscopy wider use. With recent improvements to band-stop filters the ease of processing the signal from the sample has been greatly improved and so Raman spectroscopy has started to be put to greater use.
Raman spectroscopy was used for this experiment due to its ability to not only detect the explosive compounds but also give quantifiable measurements of their concentration. The data obtained in this experiment should compliment similar measurements made using high performance liquid chromatography and also give an idea as to whether this type of spectroscopy is suitable for use on aqueous organic samples.
The partition Coefficient
The rate of degradation of the explosive compound may be limited by the rate of diffusion across E. coli's double lipid membrane. Michaelis-Menten kinetics tell us that rate of reaction is proportional to the concentration of enzyme and, at low concentrations, proportional to the compound concentration. If this compound cannot cross the cell's membrane, or the enzyme cannot diffuse outside the cell, then the effectiveness of over producing the enzyme within the cell will be reduced.
To determine the rate at which the explosive compound and enzyme will diffuse across the membrane the partition coefficient must be found. The partition coefficient of a compound is equal to the ratio of compound concentration at the surface of either side of a semi-permeable barrier. In an E. coli the barrier is the phospholipid bilayer.
To measure the partition coefficient of the phospholipid bilayer a suitable substitute will be used instead of actual phospholipid. A substitute is used because of the difficulty of setting up a bilayer in vitro from which samples could be taken from either side of. Instead the partition coefficient of the explosive compounds is measured across the boundary between olive oil and water. There is a close correlation between the partition coefficient of a compound across a phospholipid bilayer and across the olive oil water boundary found by Collander and Barlund in 1930 \cite{Partition}.
Experimental Method
A standard curve
In order for the results of any concentration measurement to be analyzed a standard curve of compound concentration versus peak area must be created. To do this the compound is mixed from a known concentration with distilled water to create solutions at a variety concentrations. For nitroglycerin concentrations of 200, 100, 66.7, 40 and 20 $\mu$gml$^{-1}$ were made up.
To measure the spectra of the compound at different concentrations, around 1 $\mu$l of the solution was pipetted onto an aluminum slide. The droplet of solution is placed under the microscope. The microscope acts as the sight for the laser, a cross hair on the microscope mounted camera output shows where the laser will become incident on the droplet.
From the literature \cite{Someone} it was found that a 785nm laser was best to detect the carbon nitrogen bonds found in nitroglycerin and TNT. The laser was set to a power of output 33 Watts, this power was recommended by the technician as a compromise between peak intensity and preventing large convection currents in the droplet from building up. Inelastic emission was scanned between wavenumber of 2000nm$^{-1}$ and 500nm$^{-1}$. Two scans were completed and added together to accentuate any peaks.
Preparation of samples and measurements in-vivo
Previous to using the Raman spectrometer samples where prepared by adding TNT and nitroglycerin to overnight cultures grown in Lysogeny Broth. Overnight cultures of Top 10 cells and Top 10 cells with coding sequences for our two degradation constructs were grown. 20 $\mu$l of TNT and nitroglycerin was added to 1978 $\mu$l of overnight culture, 2 $\mu$l of IPTG was added at the same time. 20 $\mu$l of both TNT and nitroglycerin was added to 1980 $\mu$l of Lysogeny Broth to test the background rate of degradation.
50 $\mu$l of the cultures was then pipetted out of these batches and snap frozen in liquid nitrogen. These would be used to expose any degradation that occurs after snap freezing as the concentration in these samples should be known. 50 $\mu$l samples were snap frozen at time intervals after this point. The samples were stored overnight in a -80 C$^o$ freezer before being thawed. The cells were thawed just prior to being scanned, the time between thawing and measurement was kept to a minimum with the time at which the cells were thawed and measured recorded.
After the cells are thawed they are spun down to a pellet at 800rpm for 5 minutes. 10 $\mu$l of the supernatant was pipetted off the top and stored whilst the rest of the supernatant was discarded. Measurements of both the supernatant and cells would be taken to try and determine if degradation took place inside the cells.
To take measurements of the cells roughly 2 $\mu$l of the pellet was pipetted onto an aluminum slide. The sample was spread over the slide in a small rectangle to speed up the evaporation of the remaining supernatant. Watching through the microscope convection currents caused by the remaining supernatant could be seen as ripples moving radially across the monitor. When the convection currents ceased the microscope was re-focused onto the cells and a scan between 1200 cm$^{-1}$ and 600 cm$^{-1}$ was taken for nitroglycerin. A scan between 1600 cm$^{-1}$ and 1200 cm$^{-1}$ was carried out for TNT samples.
To take measurements in the supernatant roughly 4 $\mu$l of solution was pipetted onto the aluminum slide. The microscope was focused into the droplet and the scan was started between the same wavenumber used for cells. The scan had to be completed before the droplet evaporated significantly under the laser, as this would cause the microscope to lose focus. This defocussing was the reason for using a larger volume of fluid on the slide.
Caption text
The partition coefficient
To measure the partition coefficient the aqueous explosive compounds were diluted in distilled water. 6 $\mu$l of Nitroglycerin was added to 494 $\mu$l of distilled water whilst 20 $\mu$l of TNT was added to 480 $\mu$l of distilled water. The solutions were vortexed from 30 seconds to mix. 50 $\mu$l of each of the mixtures was pipetted into a separate Eppendorf tube for use in determining the original concentration of the compounds.
450 $\mu$l of olive oil was then added to the mixtures. The oil and water combination was then vortexed for a minute, thoroughly mixing the two immiscible liquids. After around half an hour the two liquids had fully re-separated. The oil portion was pipetted off the top into a separate Eppendorf tube.
To take measurements in the water with the original concentration of explosive compound roughly 4 $\mu$l of solution was pipetted onto an aluminum slide. The microscope was focused into the droplet and a scan was completed between 2000 cm$^{-1}$ and 500 cm$^{-1}$. This was repeated for both the separated oil and water. A final scan of oil with no added compound was taken between 2000 cm$^{-1}$ and 500 cm$^{-1}$.
Results, Analysis and discussion
Figure \ref{Spectrum} shows the Raman spectrum of observed using undiluted samples of the explosive compounds. Unfortunately a literature example of the Raman spectrum from nitroglycerin was not found, as such it cannot be determined whether the spectrum seen in figure \ref{Spectrum} is that of nitroglycerin or the Propylene glycol that the compound is stored in. The nitroglycerin spectrum corresponds to the literature spectrum \cite{TNT} with its highest intensity peaks at 820$^{-1}$ and 1360 cm$^{-1}$.
The TNT spectrum has a low intensity in compared to the nitroglycerin, this is due to the nitroglycerin sample being at a higher concentration (2mgml$^{-1}$) than the TNT sample (1mgml$^{-1}$).
Caption text
The Standard curve
Figure \ref{DoublePeak} shows the normalized Raman spectrum for nitroglycerin at various concentrations between the wavenumbers of 850 cm$^{-1}$ and 750 cm$^{-1}$. The spectrum was normalized between 1200 cm$^{-1}$ and 600 cm$^{-1}$ by dividing by the average intensity between these points. It can easily be seen that the concentration is proportional to the intensity of the peaks, however the true concentration is proportional to the peak area.
Caption text
The figure \ref{DoublePeak} also shows the predicted background intensity level without the peaks. This was made by plotting a least square fitted line through measurements around the peak sites. The background intensity level increases with lower intensity peaks, this is an artifact of normalization.
Using the predicted background intensity the peak area, and thereby the concentration, can be calculated using the trapezium rule,
\begin{equation}
\text{Area} = \sum_i^{N-1} \frac{I_i+I_{i+1}}{2}(k_{i+1}-k_i).
\end{equation}
Where $I_i$ is the intensity of the $i$th measurement and $k_i$ is the wavenumber of the $i$th measurement.
The peak area is plotted against the concentration of the prepared sample in figure \ref{Standard}. A least square fitted line (Red dashed line) does not intercept the origin of the graph. This error could be due to a systematic error in pipetting the nitroglycerin into the dilutions. The stock nitroglycerin solution was quite viscous meaning that some extra nitroglycerin stuck to the outside of the pipette, this would have increased the amount of compound in each dilution.
The error in the nitroglycerin concentration attempted to account for the difficulty of pipetting correct quantities by estimating the quantity of nitroglycerin to be $\pm$ 0.5 $\mu$l. This is probably incorrect as the actual error would be biased towards greater concentrations of nitroglycerin.
Assuming that the peak height must be zero at zero concentration another line of least square fitted line was plotted, this time fixing the intercept to zero. This line will be used for determining concentrations of nitroglycerin in future measurements.
Caption text
The error in the peak area is small, it is calculated via,
\begin{equation}
\delta \text{Area} = \sigma \langle k_{i+1}-k_i \rangle \sqrt{\frac{N}{2}},
\end{equation}
were $\sigma$ is the standard deviation from the predicted background intensity of the background intensity measurements. $\langle k_{i+1}-k_i \rangle$ is the average wavenumber gap between measurements and $N$ is the number of measurements. The error in the peak area diverges from the measured area considerably for the large numbers of measurements used to calculate it.
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