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Measuring in-vivo degredation using Raman spectroscopy

Introduction

The aim of this experiment was to measure the degradation rate of trinitrotoluene (TNT) and nitroglycerin (NG) 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 TNT and NG between water and the cell membrane was 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. Inelastic scattering occurs where part of the photons energy is transferred to the vibrational state resulting in a lower energy reflected photon. 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. In the past this had been the major barrier to the wider use of Raman spectroscopy. With recent improvements to band-stop filters the ease of processing the signal from the sample has been greatly improved and as a result Raman spectroscopy has been put to greater use.

Raman spectroscopy was used for this experiment because of its ability to both detect the compounds of interest and also give quantifiable values for their concentration. The data obtained in this experiment compliments similar measurements made using high performance liquid chromatography. In addition, this also provides an early indication 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 concentration of the compound of interest (in this case TNT and NG). If this compound cannot cross the cell's membrane, or the enzyme cannot diffuse outside the cell, then the effectiveness of over producing the designed bacterial system enzyme within the cell will be greatly impaired.

To determine the rate at which TNT and NG 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.

Assembling a lipid bilayer in vitro, from which it is possible to sample on both sides, is technically challenging. Instead the partition coefficient of TNT and NG 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 [1].

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 the concentrations were 200, 100, 66.7, 40 and 20 $\mu$gml$^{-1}$.

To measure the spectra of the compound at different concentrations 1 μl of the solution was placed onto an aluminium slide. The slide was placed into the Raman 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.

A 785nm laser was chosen to detect the carbon nitrogen bonds found in nitroglycerin and TNT [3]. The laser was set to a power output of 33 Watts, this was a compromise between peak intensity and preventing large convection currents in the droplet from building up. Inelastic emission was scanned between wavenumbers of 2000nm$^{-1}$ and 500nm$^{-1}$. Two scans were completed and combined to accentuate any peaks.

Measuring degradation of TNT and NG in-vivo

After producing a standard curve the degradation rate of TNT and NG was measured in-vivo. To do this TNT and NG where added to cultures of E. coli and the concentrations of the compounds was measured over time. Overnight cultures of Top 10 cells and Top 10 cells with coding sequences both BBa_K1398001 and BBa_K1398003 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 aliquots were snap frozen in liquid nitrogen. These would be used to expose any degradation that occurs after snap freezing as the concentration in these samples was known. Further samples were snap frozen at regular time intervals thereafter 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.

Thawed cells were centrifuged at 800rpm for 5 minutes. 10 μl of the supernatant was removed. The remainder of the supernatant was discarded. Measurements of both the supernatant and cells were taken to determine if degradation occurred inside the cells.

To take measurements of the cells 2 $\mu$l of the pellet was placed onto an aluminium 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 NG. A scan between 1600 cm$^{-1}$ and 1200 cm$^{-1}$ was carried out for TNT samples.

To take measurements in the supernatant 4 $\mu$l of solution was placed onto the aluminium 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.

The partition coefficient

To measure the partition coefficient of NG 6 $\mu$l of the comound was added to 494 $\mu$l of distilled water. 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 an hour the two liquids had fully re-separated. The oil portion was removed 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 placed 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

The Raman spectrum of TNT and NG

Figure 2 shows the Raman spectrum of NG and TNT observed using undiluted samples of the explosive compounds. Unfortunately a literature example of the Raman spectrum for nitroglycerin was not found, as such it cannot be determined whether the spectrum seen in figure 2 is that of nitroglycerin or the Propylene glycol that the compound is stored in. The nitroglycerin spectrum corresponds to the literature spectrum [2] 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 to be expected as the nitroglycerin sample was at a higher concentration (2mgml$^{-1}$) than the TNT sample (1mgml$^{-1}$).

The Standard curve

Figure 3 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.

The figure 3 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 4. 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 transferring 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 NG concentration attempted to account for the difficulty of mixing the correct quantities of NG and water 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 least square fitted line was plotted, this time fixing the intercept to zero. This line will be used for determining concentrations of NG in future measurements.

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}

where $\sigma$ 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.

Measuring explosive compounds in-vivo

Figure 5 shows the normalized spectrum of E. coli cells with added nitroglycerin for four of the time points at which samples were snap frozen. The immediate difference between these spectra and the two spectra in figure \ref{Spectrum} is presence of a large fluorescence background intensity caused by the organic material in the cells.

Auto-florescence is a large problem in the analysis of organic materials using Raman spectroscopy. It is often alleviated by using a longer wavelength laser, however Raman scattering intensity is inversely proportional to the fourth order of the excitation wavelength [4] as such the sensitivity of measurements would be dramatically reduced making this option only applicable to large concentrations of sample material. Concentration of the explosive compounds were used in this experiment were approaching the limit of resolution so this option was not available.

The inset shows how the background line for measurement of peak area was determined. A least square fitted line was plotted for measurements around the peaks. The Peak area was then measured as the space between this predicted line and the peak. Only the peak centered around 840 nm was used in calculations of peak area as the lower wavenumber peak was not visible for the lowest concentration spectrum.

No nitroglycerin was detected in the supernatant. This may have been a result of centrifuging process to separate the supernatant from the cells or some unknown influx process. There was an auto-fluorescence background interference present in the supernatant spectra, probably due to organic products excreted by the cells.

Figure 6 shows the nitroglycerin concentration for four of the time points at which the samples where snap frozen. The samples were all taken in the presence of E. coli expressing the XenB enzyme. With only four plot points taken at the limit of resolution and no standard plot made (For native E. coli) it is hard to make any conclusion based solely off this result.

The method was changed on the day of the experiment so that all of the samples from a given set would be thawed at the same time, the samples would be measured in chronological order with the time at which the measurements were taken recorded. This was done to increase the time range over which the concentration could be measured. It was also done after seeing the time it took for measurements to be taken during the creation of the standard curve. Due to the difficulty of focusing the laser on the liquid droplet as it evaporated several measurements of each sample often had to be made. These repeated measurements, each around ten minutes long, would have resulted in a discrepancy between thawing times and measurement time that may have left gaps in the time scale.

Due to extensive time taken to make measurements in-vivo only four of the in-vivo samples were measured.

Partition coefficient

Figure 7 shows the spectra from the partition coefficient experiment. It can immediately be seen from the spectrum of the water after separation that the two liquids had not fully separated. This is revealed by the similarity of the water after separation spectrum and the oil spectrum. The lower auto-florescence and intensity related to olive oil peaks suggest that whilst much of the oil had separated out there were still small micro bubbles of the oil remaining.

The presence of oil in the water sample would have invalidated any results gained from this experiment, however such a small concentration of nitroglycerin was used at the outset that no peaks can be seen in either oil or water after separation anyway. The original concentration spectrum shows the maximum size of the peaks that would be expected, these are simply below the limit of resolution in the other spectra.

It is interesting to note that the auto-florescence increases in the oil samples after separation. Perhaps the boundary between water and oil creates a strong reflection, this may also explain the apparent magnitude of the oil remaining in the separated water sample.

Summary

References

1. COLLANDER, R., AND BARLUND, H.: Acta Botan. Fennica 11, 1 (1930)
2. Trace level detection and identification of nitro-based explosives by surface-enhanced Raman spectroscopy, S. Botti,* S. Almaviva
3. Explosive Detection using the D3 Klarite SERS technology, D3 Technologies Ltd.
4. Fluorescence Background Problem in Raman Spectroscopy, Shan Yang and Ozan Akkus

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